CN101595129B - The crystal structure of THSR acceptors - Google Patents

Abstract

The present invention relates to a kind of crystallizable composition containing TSHR polypeptides, is related to a kind of crystal containing TSHR polypeptides and is related to the application relevant with TSHR.

Description

The crystal structure of THSR acceptors

Technical field

The present invention relates to thyrotropin receptor --- also referred to as thyrotropin receptor (TSHR) and anti-with TSHR The autoantibody answered, especially by the interaction between the TSHR of X-ray crystallography measure and this autoantibody.

Background technology

Thyroid-stimulating hormone is that thyrotropic hormone (TSH) is a kind of pituitrin that thyroid function is adjusted by TSHR (Szkudlinski MW, Fremont V, Ronin C, Weintraub 2002Thyroid-stimulating hormone and TSHR structure-function relationships.Physiological Reviews 82：473-502). TSH is attached to TSHR and triggers acceptor to send signal, and it promotes thyroid hormone thyroxine (T4) and triiodo thryonine (T3) formation and release.There is a kind of feedback mechanism to be related to T4 and T3 level and the rush first shape secreted by hypothalamus in circulation Hormone releasing hormone (TRH), this mechanism control TSH release, TSH control thyroid gland to stimulate and thyroid gland in serum in turn The level of hormone.

TSHR is a kind of acceptor of G-protein coupling, and it contains three domains：Rich leucine domain (LRD), cutting knot Structure domain (CD) and membrane spaning domain (TMD) (Miguel R, Sanders J, Jeffreys J, Depraetere H, The Analysis of the of Evans M, Richards T, Blundell TL, Rees Smith B, Furmaniak J 2004 thyrotropinreceptor-thyrotropin interaction by comparative modeling.Thyroid 14：991-1011).TSHR and other glycoprotein hormone receptors are luteinizing hormone receptor (LHR) and Follicle Stimulating Hormone Receptors (FSHR) there is amino acid and structural similarity.Compound FSHR structure is 2.9 with its part (i.e. FSH)Point (the Structure of human follicle- of Fan QR, HendricksonWA 2005 are revealed under resolution stimulating hormone in complexwith its receptor.Nature 433：269-277).

Many data explanation of this area, (AITD, one kind influence world wide to some AITD patients The most common autoimmune disease of interior different crowd) have with TSHR reaction autoantibody (Rees Smith B, The Autoantibodies to the thyrotropin of McLachlan SM, Furmaniak J 1988 receptor.Endocrine Reviews 9：106-121).In most cases, these autoantibodies are combined simultaneously with TSHR TSH effect is imitated, so as to stimulate thyroid gland to produce high-caliber T4 and T3.These autoantibodies are referred to as thyroid itself Antibody is TSHR autoantibodies (TRAb), and it has stimulating activity i.e. TSH agonist activities.The generally controllable thyroid gland of feedback mechanism Function, but in the presence of thyroid autoantibody and with hyperthyroidism shape (thyroid gland in serum Hormonosis) patient in it is no longer valid.This illness is referred to as hyperthyroidism or GraveShi diseases.Have to stimulate in some patients and live Property TRAb be considered as in retrobulbar tissue with TSHR interact, and cause GraveShi disease eye conditions.

Autoantibody in some AITD patients is combined with TSHR, so as to prevent TSH from being combined with the acceptor, but is not pierced Swash the ability of TSHR activity, the autoantibody of these types is referred to as having the blocking activity i.e. TRAb of TSH antagonistic activities.

When TSHR autoantibodies are present in high concentration in the serum of pregnant woman, it may pass through placenta and cause neonate's first High (in the case of excitant autoantibody) or neonatal hypothyroidism (in the case of blocking property autoantibody) (Rees Smith B, McLachlan SM, Furmaniak J1988 supra).

Human monoclonal autoantibody as the strength thyroid factor (hMAb TSHRl) is in international patent application Had a detailed description in WO2004/050708A2.HMAb TSHRl binding site is found to be located at TSHR richness leucine domains (LRD) surface, and it is widely overlapping with TSH binding sites.However, TSH or hMAb TSHRl binding pocket (binding Pocket) there is certain conformation, include the TSHR folded discontinuity zone.HMAb TSHRl binding site it is thin The minutia of feature, especially contact amino acid important in TSHR with hMAb TSHRl interactions is saved, to being intended to carry It is very important in the research of diagnosis and the management of the high disease relevant with TSHR autoimmune responses.

International patent application WO 2006/016121A disclose a mutation T SHR system for including at least one point mutation Agent, it can be used for patient serum stimulating TSHR autoantibodies in the patient body fluid sample that is screened, patient serum blocking TSHR autoantibodies and thyrotropic hormone carry out differential screening and identification.Described in international patent application no WO2006/016121A Invention provide useful information on TSHR regions, the TSHR regions for including hMAb TSHR 1, there is TSHR Multiple Antibodies including the mouse monoclonal antibody (9D33) of blocking activity and critically important with TSH interaction.However, i.e. Be best experimental study (such as those researchs for being related to TSHR mutation described in WO2006/016121 A) also without Method obtains the details of the interaction on atomic level between the amino acid in amino acid and hMAb TSHRl in TSHR.

The present invention is based on by TSHR LRD fragments (participation forms TSH and hMAb TSHRl binding pocket) and hMAb TSHRl Fab fragments formed compound preparation.For convenience, the hMAb TSHRl preparations described in this specification are referred to as M22. M22 IgG can be bought from RSR companies.TSHR fragments (TSHR260) comprising amino acid/11-260 and hMAb TSHRl (M22) Fab The compound of fragment is referred to as TSHR260-M22.TSHR260-M22 is purified, concentrate and crystallized.Number derived from X-ray diffraction According to the structure for disclosing TSHR260, as described herein.Document in the past is described 1.65Resolution ratio under disclose M22 Fab structure (Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Kiddie A, Brereton K, Premawardhana LDKE, Chirgadze DY, Nunez Miguel R, Blundell TL, The Characteristics of a humanmonoclonal of Furmaniak J, Rees Smith B 2004 autoantibody to the thyrotropin receptor：sequence structureand function.Thyroid 14：560-570).By the M22 in compound compared with uncombined its structure of M22.Then in original Interaction between sub horizontal accurate research TSHR260 and M22.

Now, it is intact through highly purified TSHR preparations that itself TSH and TRAb binding activity can not also be obtained.Ability Purified TSHR preparations described in domain are partial denaturations, and are not reaching to the enough purity and homogeneity needed for crystallization.

The content of the invention

One aspect of the present invention provides a kind of crystallizable composition, comprising TSHR polypeptides, that is, contains and comes from rush first The polypeptide of the adjacent amino acid of the primary sequence of shape adrenoceptor.

Another aspect provides a kind of crystal for including TSHR polypeptides.

Another aspect provides a kind of crystallizable comprising TSHR polypeptides and TSHR binding entities (entity) Compound.

The advantages of this compound, is that TSHR binding entities can stablize the TSHR polypeptides.The present invention also provides and produces bag The method of the crystallizable compound of polypeptide containing TSHR and TSHR binding entities, wherein the TSHR binding entities are more to the TSHR Peptide has relatively high compatibility compared with to TSH, and can stablize the TSHR polypeptides.A kind of suitable TSHR binding entities are M22.TSHR joints can be used for carrying out it when TSHR polypeptides produce stabilization.For example, encode the DNA of TSHR- polypeptides in expression When TSHR polypeptides are produced in the cell such as insect cell of construct, TSHR joint such as M22 Fab can be added to the cell, so that By its " capture " and stably when TSHR polypeptides are secreted.

Another aspect provides a kind of DNA construct that TSHR polypeptides are encoded by expression is more to produce TSHR The method of the compound of peptide and TSHR joints, methods described include expression TSHR polypeptides and addition TSHR joints with stably excreting TSHR polypeptides.

Another aspect provides it is a kind of by expression encode be connected to TSHR binding entities such as M22 TSHR it is more The DNA construct of peptide produces the method for the compound of TSHR polypeptides and TSHR joints, and methods described is connected to including expression The TSHR polypeptides of TSHR binding entities.

Another aspect provides a kind of cocrystallization (co- comprising TSHR polypeptides and TSHR binding entities crystal)。

TSHR polypeptides preferably include mammalian TSHR sequences.Preferably, the mammalian cell is people source, but It is to also contemplate for using orangutan, cercopithecus aethiops, macaque (rhesusmonkey), dog, cat, pig, horse, ox or cavy.Preferably, institute Stating TSHR polypeptides includes at least a portion of TSHR richness leucine domains, most preferably human sequence.Preferably, it is described TSHR polypeptides include the amino acid 22-260 of Wild type human's TSHR sequences.TSHR polypeptides in compound of the present invention can include TSHR full wild-type sequences.Or the TSHR polypeptides may include the mutant nucleotide sequence of wild-type sequence.For example, wild type sequence The specific amino acids of row can be replaced by other amino acid.These substitutions are conservative, i.e., an amino acid is had by another The amino acid replacement of similar quality.TSHR binding entities can be an antibody or one part.One suitable antibody is one Autoantibody or one part or the TSHR binding entities from this autoantibody.Suitable antibody includes monoclonal antibody.One Individual suitable antibody moiety is M22 Fab.

Another aspect provides a kind of cocrystallization of the TSHR polypeptides comprising crystal form, the TSHR has Fig. 9 a or the 9b location data (co-oridinate) determined by X-ray crystallography.

Analysis to cocrystallization of the present invention provides the M22 compound with TSHR260 2.55Atom under resolution ratio is determined Position data and structure factor.This horizontal confidence level can only be from the compound to being formed by the two molecules (TSHR and M22) Structure carry out at high resolutions X-ray crystallography analysis in obtain.This is beneficial because with prediction for reference to and The other method of the amino acid wanted of overstating is compared, and the crystal structure analysis only carried out to the compound between two molecules can be right Interaction (the interatomic distance for including the residue of participation) is identified.In addition, the complexity between acceptor and part is mutually Effect can only obtain from crystal structure.

The information that compound M22 crystal structure is provided with TSHR is wondrous.Especially, can not obtain in the past described Information, such as modeling and mutating experiment research can only provide on participate in TSHR and TSH between and TSHR and TSHR itself The basic clue of the amino acid of the interphase interaction of antibody.All these early stage researchs are shown in anti-to TSH and TSHR itself Exist between the TSHR binding sites of body very big overlapping.Also clearly illustrate that TSHR and FSHR are closely related in structure.

Show the complicated journey of whole of interaction of the TSHR autoantibodies (such as M22) between TSHR however, coming to nothing The true detailed construction of degree or TSHRLRD.

Another aspect of the present invention provides machine readable data storage medium, and the medium is by more with Fig. 9 a or 9b TSHR The relevant data encoding of at least a portion location data of peptide ammino acid or its homologue.Preferably, the data include all Fig. 9 a or 9b TSHR polypeptide amino acid location datas.

Another aspect of the present invention provides the calculating for being used to show TSHR polypeptides or the tomograph of its homologue Machine system, wherein the homologue has 0 apart from backbone atomsTo 4Root-mean-square-deviation, the computer system includes The data storage medium of institute data storage corresponding diagram 9a or 9b TSHR polypeptide amino acid location datas.The computer system is also It may include that the data of the location data of the corresponding chemical entities to be interacted with TSHR polypeptides or its homologue of institute's data storage are deposited Storage media.

Preferably, the computer system is for the TSHR polypeptides or its homologue of offer and chemical entities interaction Tomograph.The chemical entities can be an antibody or one part.Preferably, the antibody moiety is M22 Fab.

The computer system may include the display for showing TSHR polypeptide tomographs.Preferably, the meter Calculation machine system is used to show the chemical entities to interact with TSHR polypeptides or its homologue.

Another aspect of the present invention provides the electronic image of the three-dimensional structure of TSHR polypeptides.Preferably, the TSHR polypeptides Rich leucine domain including mankind TSHR.It is highly preferred that described image at least shows mankind TSHR amino acid 30-257. Another aspect of the present invention provides TSHR polypeptides and its electronic image of the three-dimensional structure of antibody or their part.

Another aspect provides a kind of at least one amino acid phase interaction identified with TSHR or its homologue The method of chemical entities, the three-dimensional structure of the TSHR or its homologue are by the computer according to previous aspect of the invention Image or the electronic image displaying that system is provided.The chemical entities can be TSHR activators or antagonist.With TSHR ammonia At least one chemical entities by forming interaction of hydrogen bond in base acid K129, E107, K58 and Y185 can be identified.Or Person or in addition, at least one formation Van der Waals force phase interaction with TSHR amino acid residues R255, R80, Kl29, R38 and Kl83 Chemical entities can be identified.Besides or furthermore, with TSHR amino acid residues D151, K58, Kl29, R80, K209 and Kl83 The chemical entities of at least one formation electrostatic interaction can be identified.Besides or furthermore, with TSHR amino acid residues K209 It can be identified by the chemical entities for forming ion Thermodynamic parameters.

Another aspect of the present invention provides a kind of side for identifying and may interfere with the chemical entities that autoantibody is combined with TSHR Method, methods described include identification and interacted with the positively charged ridge of the height in TSHR richness leucine domain N-terminals concave surface Chemicals.The autoantibody is probably that thyroid autoantibody or TSH antagonists have the TSH of blocking activity certainly Body antibody.Suitable chemical entities can be with least one interaction in following TSHR amino acid：R38、K58、R80、Hl05 And Kl29.

Another aspect of the present invention provides a kind of identification and may interfere with the chemical entities that autoantibody is combined with TSHR Method, methods described include at least being filled substantially with being become by M22 is high with computer or the electronic image identification of previous aspect of the present invention The method of the chemical entities of negatively charged cavity on the M22 surfaces that area H1, H2 and H3 (Fig. 5) are formed.The autoantibody can Can be the TSH autoantibodies that thyroid autoantibody or TSH antagonists have blocking activity.

Usual this method includes identification and at least destroys what TSHR residues R255 was combined with thyroid autoantibody substantially Chemical entities.Besides or furthermore, methods described, which includes identification, destroys mankind TSHR residues K209 and thyroid autoantibody knot The chemical entities of conjunction.

In the inventive method, certain chemical entities and the possible interaction of other acceptors, the chemoreceptor are determined It is identified as to be combined with TSHR polypeptides, or may interfere with and the combination of the TSHR polypeptides of TSHR joints.It is a kind of possible mutual The form of effect is to combine.Other acceptors may include Follicle Stimulating Hormone Receptors or luteinizing hormone receptor.

Another aspect provides a kind of method of detection TSHR autoantibody, including compare supposition itself Antibody and the combination with the chemical entities and TSHR polypeptides of the more peptide interactions of TSHR is identified as by the inventive method.

The present invention is favourable in many aspects, because it causes technical staff to design specificity and prevent thyroid certainly The new pharmaceutical compositions that body antibody is combined with TSHR, so as to provide the new treatment to autoimmune disease such as GraveShi diseases.This hair The bright new pharmaceutical compositions for also causing technical staff to design the tissue that differential stimulus contains TSHR.

Another aspect of the present invention provides the chemical entities identified by the method for previous aspect of the present invention.The present invention's is another On the one hand the compound for including at least one this chemical entities is provided.Another aspect of the present invention provides and includes this compound With a kind of pharmaceutical composition of pharmaceutical acceptable carrier.

This pharmaceutical composition is applied to the treatment of AITD.Or this pharmaceutical composition can fit Treatment for GraveShi diseases.A kind of pharmaceutical composition for being used to stimulate the tissue containing TSHR is also provided.

Chemical entities provided by the invention are likely to be suited for detecting TSHR autoantibody.

Another aspect provides the foregoing aspects of chemical entities of the present invention or compound to prepare treatment itself Purposes in the medicament of autoimmune thyroid disease.

Another aspect provides the foregoing aspects of chemical entities of the present invention or compound to prepare treatment Grave Purposes in the medicament of family name's disease.

The pharmaceutical composition of the present invention includes any compound of the present invention and its pharmaceutically acceptable salt, and any Pharmaceutically acceptable carrier, adjuvant or carrier.Available for the pharmaceutically acceptable carrier of pharmaceutical composition of the present invention, adjuvant and Carrier include but is not limited to ion-exchanger, aluminum oxide, aluminum stearate, lecithin, haemocyanin (such as human serum albumins), Buffer substance (such as phosphate), glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acid partial glyceride mixtures, Water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, Ludox, three silicic acid Magnesium, polyvinylpyrrolidone, cellulosic material, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene polyoxy Propylene block copolymer, polyethylene glycol and lanolin.

The administration of the pharmaceutical composition of the present invention can by oral, parenteral, spraying suction, part, eye drops or eye ointment, Rectum, nasal cavity, oral cavity, vagina or implantation holder.Our preferred oral or injection administrations.The pharmaceutical composition of the present invention can contain There are any traditional non-toxic pharmaceutical acceptable carrier, adjuvant or carrier.Term used herein is " parenteral " include it is subcutaneous, Intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, breastbone be interior, in intrathecal, focus and intracranial injection or infusion techn.

Described pharmaceutical composition can be the form of sterile injectable preparation, such as the water or oil suspension of sterile injectable.This Kind suspension can be made according to known technology using suitable dispersant or wetting agent (such as Tween 80) and suspending agent.It is sterile can Injection reagent may also be sterile injectable solution or suspension made of nontoxic parenteral acceptable diluent or solvent, such as It is dissolved in the solution form of 1,3-BDO.Workable acceptable carrier and solvent are mannitol, water, ringer's solution and waited Ooze sodium chloride solution.In addition, sterile fixed oil is often used as solvent or suspending medium.Appoint for this purpose, can be used What gentle fixed oil, include the monoglyceride or double glyceride of synthesis.Aliphatic acid such as oleic acid and its glycerol derivatives can For preparing injectable agent, as natural pharmaceutically acceptable oils (such as olive oil or castor oil) especially its polyoxyethanyl Change form is the same.These oil solutions or suspension are also containing long-chain alcohol diluents or dispersant such as Ph.Helv or similar alcohol.

The pharmaceutical composition of the present invention can be administered orally with any oral acceptable medicine type, including but not limited to glue Capsule, tablet and water slurry and solution.For tablets for oral use, conventional carrier includes lactose and cornstarch.Generally also add Enter lubricant such as magnesium stearate.For capsule for oral administration form, useful diluent includes lactose and dry cornstarch.Work as water When suspension is used for oral, its active component is combined with emulsifying agent and suspending agent.If desired, it can also add some sweeteners And/or flavor enhancement and/or pigment.

The present invention pharmaceutical composition can also rectally be administered with suppository form.Can by by the present invention compound Mixed with suitable non-irritating excipient to prepare these compositions, the excipient is at room temperature for solid-state but in rectum temperature It is liquid that degree is lower, thus can be dissolved in the rectum so as to discharge active component.This material includes but is not limited to cocoa butter, honeybee Wax and polyethylene glycol.

When required treatment includes region or the organ that local drug delivery is accessible to, the part of pharmaceutical composition of the present invention is given Medicine is particularly useful.For topical cutaneous administration, described pharmaceutical composition should be made containing the work being suspended or dissolved in carrier The suitable ointment of property component.Carrier for the compounds of this invention local application include but is not limited to mineral oil, liquefied petroleum, White oil, propane diols, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Or described pharmaceutical composition can be made into containing The suitable lotion or creme for the reactive compound being suspended or dissolved in carrier.Suitable carrier includes but is not limited to mineral Oil, Arlacel-60, polysorbate60, cetyl esters wax, palmityl alcohol, 2- octyldodecanols, benzene first Alcohol and water.The pharmaceutical composition of the present invention suitably enema forms can also be used for lower digestive tract.Present invention additionally comprises part Transdermal patches.

The pharmaceutical composition of the present invention can be administered in the form of nasal spray or inhalant.These compositions are according to medicine Prepared by technology known to formulation art, and phenmethylol or other suitable preservatives, the absorption rush for strengthening bioavilability can be used Enter agent, fluorocarbon/or the cosolvent understood in the industry with other or dispersant to be prepared as saline solution.

The pharmaceutical composition of the present invention can be administered in the form of eye drops, eye cream or eye ointment.For eye drops or eye cream, Suitable excipient includes but is not limited to water, tonicity modifying agent (such as sodium chloride), preservative (such as benzalkonium chloride) and/or buffer (such as sodium dihydrogen phosphate-water and/or anhydrous dibasic sodium phosphate).For eye ointment, suitable excipient includes but is not limited to albolene And/or yellow petroleum jelly, lanolin and/or atoleine.

Measure can be designed the invention enables technical staff and assesses the new method of the autoantibody for TSHR.It is some existing Measure and assess the existing TSHR preparations produced by recombinant DNA technology used in the method for TSHR autoantibody It is very expensive.In addition, the TSHR preparations used at present are not soluble in water, it is necessary to the presence of detergent, thus it is relative " coarse ", Mixture that i.e. they contain other albumen and the mixture for representing denaturation acceptor and consistency acceptor.The TSHR polypeptide groups of synthesis Compound can be found (without 7 transmembrane regions that TSHR is complicated, you can be dissolved in water and be easily handled) according in compound of the present invention Interaction be configured to have TRAb binding properties.This composition can be used for the sensitive isotope established or non-same position Element is tested to measure TRAb.These new methods can be based on to M22 (or another TSHR monoclonal autoantibodies or TSHR monoclonals from The mixture of body antibody or from their compositions) with reference to suppression or can based on the composition directly in conjunction with. The composition can mark or be conjugated to a variety of reagents known in the art (with isotope marks or nonisotopic labels).It is described Composition can be coated on solid support (pearl, plank, test tube) or in solution in precipitation determines.

On the contrary, the TSHR binding compositions of synthesis may be configured to suppressive experiment, to substitute M22, (or other TSHR are mono- Clonal antibody) or TSH.Production and purifying currently used for the M22 IgG or TSH of these experiments is all relatively expensive.By enzymic digestion Obtained M22 IgG fragments (such as Fab or F (ab ')₂) be not as stable as IgG, production is expensive, and it is with isotope or heterotope It is not disposable during mark mark.M22 IgG or TSH TSHR binding compositions be may replace with isotope or nonisotopic labels Can be more stable when marking or be conjugated to other reagents.

Moreover, the combination of the synthetic composition and synthesis TSHR splice combinations things of TSHR combination epitopes can cause foundation to be suitable to Sensitive, the easy production of automation, easily use, economic TRAb experiments.In addition, it can design and test the invention enables technical staff The mutation of specific amino acids in M22 and TSHR, this can result in a finding that the amino acid to be played a crucial role to receptor activation.

Moreover, the present invention can help technical staff to understand TSHR (participating in thyroid gland regulation and control) and Follicle Stimulating Hormone Receptors (FSHR；Participate in reproduction) between the similarities and differences.TSHR and FSHR is the acceptor of height correlation, and they have similar structure, tied respectively Close with common hormone subunit (α subunits) and show the part TSH and FSH of very big structure similitude.However, TSHR and FSHR tools There is different functional activities；TSHR participates in thyroid gland regulation and control (being played an important role to general metabolism), and FSHR participates in reproduction.This A little acceptors are very high to the specificity of their corresponding hormones, studies have reported that even if using very high molar concentration hormone, also only There is low-level cross reactivity.Now, technical staff can automatically study both acceptors first, compare their structure With the binding mechanism with their corresponding parts.For example, as disclosed herein, to the comparison (Fan of TSHR structures and FSHR structures The infra of ＆ Hendrickson 2005) significant similitude is shown (in C_αThere was only 1.1 on atomrmsd).Moreover, FSHR and FSH binding mechanism is closely similar with TSHR's and M22.Now, the binding mechanism that can study TSH and TSHR (uses Obtainable structure and methods known in the art), and it is compared with the binding mechanism with FSHR-FSH.Pass through this area Known method is recognizable to cause its each specific TSH or FSH amino acid, and the evolutionary process of the analysis hormone.It is right The further understanding of this 2 kinds closely related receptor-hormones interactions can help technical staff's design have to TSHR or FSHR high degree of specificity and the part that unwanted cross reaction does not occur.Such part can be used for the tune of reproductive system Control and the control for thyroid function.

The present invention may also aid in technical staff understand can promote the exploitation of specific TSH autoantibodies and universal autoantibody and Caused amynologic mechanism.

In addition, present invention also offers the water-soluble TSHR peptide compositions of synthesis.The peptide composition may include people Class TSHR amino acid 22-260.

The data that TSHR260-M22 crystal structure provides can help to design binding molecule (such as polypeptide), and it imitates TSHR The binding site of upper M22 (and TSHR autoantibodies with similarity surface and binding property).This binding molecule can be with conjunction Suitable holder coupling, for removing thyroid autoantibody.Similar method has been used for oneself of removing acetylcholinergic receptor Body antibody (GuoC-Y et al, Journal of Immunological Methods, 2005,303, pp 142-147).Cause This, another aspect of the present invention offer is a kind of to remove these thyroid-stimulating hormone from the sample containing serum thyrotropin receptor antibody The method of receptor antibody particularly thyrotropin receptor autoantibody, methods described, which includes offer one, to be included or imitates to promote first M22 or have and the combination of the thyrotropin receptor autoantibody of the similar surfaces of M22 and binding property on shape adrenoceptor The binding molecule in site；The sample is contacted with the binding molecule again so that serum thyrotropin receptor antibody with it is described Binding molecule is combined and removed from sample.Suitable sample may include the trouble containing high-caliber thyroid autoantibody Person's serum.To promote to be coupled, the binding molecule can with neutral protein or other do not influence the mark that TSHR autoantibodies are combined Fusion.Such as maltose-binding protein can be used, as disclosed in Guo C-Y (2005) et al, supra.The binding molecule It can directly be coupled with solid support such as agarose or resin or be coupled by a this mark.Then can make described containing height By immunosorbent, (one is similar to plasma exchange or plasmapheresis to the patients serum of horizontal thyroid autoantibody Process), then the serum of removing TSHR autoantibodies is returned to patient.This causes thyroid gland excessively to pierce efficiently and quickly to remove The autoantibody of sharp clinical symptoms provides a good opportunity.This is even more important to thyroid crisis.Moreover, removed from circulation TSHR autoantibodies can prevent them from being combined with the TSHR of tissue after eye (or the outer site of other thyroid glands), so as to provide control The effective ways of Grave illness in eye (or circumscribed myxedema) several cases.TSHR itself is removed from the circulation of pregnant woman Antibody can prevent the autoantibody from being passed through across placenta, and protect Fetal Thyroid Gland from the biology effect of autoantibody.Cause This present invention also provide it is a kind of treat the method for this disease as described above by autoantibody is removed, methods described include from The step of thyroid autoantibody being removed in patient with this disease or the sample obtained from this patient.

Brief description of the drawings

The product and method of the present invention are described in an exemplary manner referring now to accompanying drawing 1-10, wherein：

Fig. 1 a represent to enter the TSHR260 expressed in High Five cells in the case where being not present or M22 IgG being present Capable western blot analysis.Fig. 1 b represent the culture supernatant to being obtained from the High Five cells for expressing corresponding TSHR constructs The western blot analysis that SHR277, C-del TSHR and TSHR260 are carried out before and after partial purification in liquid.Fig. 1 c are represented in High The western blot analysis that the C-del TSHR of Five cells expression are carried out in partially purified different phase；

Fig. 2 a and b show the result of HPLC gel filtrations；Fig. 2 c are after TSHR 260-M22 Fab compounds produce The photo of SDS-PAGE electrophoresis results；

Fig. 3 is the 2F for representing TSHR 260-M22 Fab compound combination interfaces₀-F₀The perspective view of electron density map Figure；

Fig. 4 show the TSHR LRD shown with JOY forms secondary structure (Mizuguchi K, Deane CM, The JOY of Blundell TL, Johnson MS, Overington JP 1998：proteinsequence-structure representation and analysis.Bioinformatics 14：617-623)(the TSHR LRD amino acid sequence is SEQ ID NO：14)；

Fig. 5 is a series of figure of expression M22 Fab and TSHR interactions：

Fig. 5 a represent the molecular surface of TSHR-M22 Fab compounds；

Fig. 5 b are the opening visual angle figures of the surface region, and the distance that it is protruded and the residue of mark is mutual is all 4Within；

Fig. 5 c are shown with the hypervariable region for the M22 Fab that different shades is prominent and mark is dated；

Fig. 5 d represent the electrostatic potential surface of TSHR-M22 Fab compounds；

Fig. 5 e are the residue lists of M22 Fab hypervariable regions；

Fig. 6 a are the imaging importings of FSHR-FSH composite structures and TSHR-M22 Fab composite structures；

Fig. 6 b show the image that Fig. 6 a turn 90 degrees to obtain along vertical axis dextrorotation；

Fig. 6 c be TSHR and FSHR according to the structure-based sequence alignment of JOY forms (Mizuguchi K, Deane CM, Blundell TL, Johnson MS, Overington JP 1998JOY：protein sequence-structure representation and analysis.Bioinformatics 14：617-623)(the FSHR amino acid sequence is SEQ IDNO：15)；protein sequence-structure representation and analysis.Bioinformatics 14：617-623)(the FSHR amino acid sequence is SEQ IDNO： 15)；

Fig. 6 d are the space multistory ball images that TSHR LRD contact surface with M22 Fab and FSHR LRD with FSH.Participate in The amino acid of interaction is represented with Dark grey；

Fig. 7 is the schematic diagram by the amino acid residue of TSHR-M22 Fab compound interfacial interactions；

Fig. 8 be observed at the antagonist-receptor interface of TSHR-M22 Fab compounds direct interaction () perspective view figure；

Fig. 9 is derived from the location data table of following crystallography experiments, wherein：

FigureResolution list；

Figure(chain A=mankind's thyroid autoantibody M22 light chains, the chain B=mankind promote resolution list Thyroid autoantibodies M22 heavy chains, chain C=mankind thryoid receptor (TSHR), fragment=full asphalt mixture domain (section 22-260).In M22 location datas, the light chain leucine of position 1 and the heavy chain threonine of position 131 are shown as alanine. In TSHR location datas, the glutamic acid of position 34,35, the aspartic acid of position 36；The phenylalanine of position 37, position 42 it is bright Propylhomoserin, the arginine of post-11.2 are shown as alanine.It is because lacking electronics that these residues, which are modeled as alanine or glycine, Density)；

Figure 10 a show TSHR consensus amino acid sequences (SEQ ID NO：16) (acquisition P16473,

http://www.ncbi.nhn.nih.gov/entrez/viewer.fcgiDb=protein＆val= 62298994)；

Figure 10 b are shown in M Misrahi, H Loosfelt, M Atger, S Sar, AGuiochon-Mantel, E Milgrom.Cloning, sequencing and expression ofhuman TSH receptor.Biochem Biophys Res Commun 1990 166：Sequence (the SEQ ID NO identified in 394-403：17) (acquisition M32215, http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgiDb=nucleotide＆val=307524)；

Figure 10 c are shown in AL Frazier, LS Robbins, PJ Stork, R Sprengel, DLSegalofi ζ RD Cone.Isolation of TSH and LH/CG receptor cDNAs fromhuman thyroid：regulation by tissue specific splicing.MoI Endocrinol1990 4：Sequence (the SEQ ID identified in 1264-1276 NO：18) (acquisition M73747, http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgiDb= Nucleotide＆val=903759)；

Figure 10 d are shown in Y Nagayama, KD Kaufman, P Seto, B Rapoport.Molecular Cloning, sequence and functional expression of the cDNA forthe human thyrotropin receptor Biochem Biophys Res Commun 1989 165：The sequence identified in 1184-1190 Arrange (SEQ ID NO：19) (acquisition M31774, http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi Db=nucleotide＆val=340003)；

Figure 10 e show a control sequence (SEQ ID NO：20) (acquisition M73747, http:// www.ncbi.nlm.nih.gov/entrez/viewer.fcgiDb=nucleotide＆val=64085120)；

And Figure 10 f show sequence (the SEQ ID NO provided in the 509A of EP 0 433：21).

Embodiment

TSHR260 is produced in insect cell

Using total length mankind TSHR as template (Oda Y, Sanders J, Roberts S3 Maruyama M, Kato R, Perez M, Petersen VB, Wedlock N, Furmaniak J, Rees Smith B1998 Binding characteristics of antibodies to the TSH receptor.Journal of Molecular Endocrinology 20：233-244), with 5 '-cactgcaggatccaaatgaggccggcggacttg-3 ' (SEQ ID NO：And 5 '-cagtcctctagattatcagtgatggtggtggtgatggttaagagtccaggtgtttc ttgctat-3 ' 1) (SEQ ID NO：2) primer (Sigma Genosys) amplification TSHR260 construct (mankind's TSHR ammonia shown in code pattern 10a Base acid 1-260), a BamHI restriction site is added in N-terminal, 1 Amino acid linker (asparagine) is added in C-terminal With a 6 histidine-tagged, terminator codons and an XbaI restriction site.PCR reaction carry out 25 times 95 DEG C 1 minute, 50 DEG C of 1 minute and 72 DEG C of circulations of 1 minute, are then cloned into pFastbacl with BamHI and Xbal restriction sites by TSHR 260 In (Invitrogen, UK), and confirm DNA sequence dna (Sanger F, Nicklen S, Coulson with Sanger Coulson methods AR 1977 DNA sequencing with chain terminating inhibitors.Proceedings of the National Academy of Sciences of the USA 74：5463-5467).Restructuring rod granule DNA passes through Bac to Bac baculoviral table systems (Invitrogen, UK) are made.In short, 1ng (5 μ L) pFastBacl-TSHR 260 is transfected To the D H10Bac cells containing rod granule (baculovirus shuttle vector) and helper plasmid of 100 μ L maximal efficiencies In (Invitrogen, UK).After ice bath 30 minutes, by the cell in 42 DEG C of heat shocks 45 seconds, then divide in cooled on ice 2 Clock, add 900 μ L SOC culture mediums (Invitrogen, UK).Test tube is shaken into culture 4 hours at 37 DEG C and then trained with SOC 10 times of serial dilutions of base are supported, are inoculated in containing 50 μ g/mL kanamycins, 7 μ g/mL gentamicins, 10 μ g/ml tetracyclines, 100 μ G/mLX-gal (Promega, UK) and 40 μ g/mL isopropyl-β-D-thiogalactosides (IPTG) LB agar plates (pancreas egg White peptone lOg/L, yeast extract 5g/L, NaCl 10g/L and 20g/L agar) on, then cultivate at 37 DEG C 48 hours with carry out it is blue/ White selection.White colonies are cultivated, with plasmid midi kits (Qiagen, UK) Prepare restructuring DNA and use PCR Confirm the presence of TSHR260DNA in restructuring rod granule.

The restructuring rod granule DNA is transfected into Sf-9 insect cells (Invitrogen, UK), added with 10% (v/ V) cultivated in the TC100 culture mediums (Invitrogen, UK) of hyclone and 7 μ g/ml gentamicins, to obtain and expand Recombinant baculovirus original seed.Centrifuged by 500g and harvest virus stocks from SF-9 cultures within 5 minutes and be stored in supernatant 2-8℃.Illustrated according to manufacturer to titrate all virus with BacPAK baculoviral rapid titrations kit (BD Clonetech) Original seed.

In addition, two TSHR constructs being used in insect cell system expression are prepared for as also described above.It is complete with the mankind Long TSHR is template, uses primer：5’-caggaaacagctatgac-3’(SEQ ID NO：And 5 ' 3)- gctactcgagctagtggtggtggtggtggtgaaggtcagcccgtgtgaggtgaaggaaactcaag-3’(SEQ ID NO：4) (Sigma Genosys) expands TSHR277 constructs (mankind TSHR amino acid/11s -277 shown in code pattern 10a), institute State construct and be added to a 6 histidine-tagged, terminator codons and an Xhol restriction site in C-terminal.The PCR is anti- Should carry out 25 times 95 DEG C 1 minute, 40 DEG C of 1 minute and 72 DEG C of circulations of 1 minute, then will with BamHI and XhoI restriction sites TSHR 277 is cloned into pFastbacl (Invitrogen, UK), and confirms DNA sequence dna with Sanger Coulson methods (the DNA sequencing with chainterminating of Sanger F, Nicklen S, Coulson AR 1977 inhibitors.Proceedings of the National Academy of Sciencesof the USA 74：5463- 5467).TSHR260 restructuring rod granule DNA is made and the presence of restructuring rod granule is confirmed with PCR.Restructuring rod granule DNA is transfected into Sf-9 insect cells, virus stocks and the titration of TSHR260 constructs are prepared as described above.

C-del TSHR constructs are also prepared for, the construct coding is except coding TSHR amino acid 313-353 sequence TSHR amino acid/11s -410 outside (by being deleted from sequence) (TSHR amino acid is as shown in Figure 10 a).In addition, the TSHR C- Three mutation i.e. R312E, E358T and E360T that del constructs contain to prevent proteolytic cleavage and introduce.The structure Build four independent stages of preparation point of body.First double mutation E358T/E360T are introduced by template of total length TSHR.As existed before Two different PCR reactions (PCR1 and PCR2) are established described in WO2006/016121A.PCR1 reactions use T7 promoter primers 5’-taatacgactcactataggg-3’(SEQ ID NO：5) and for mutation " reverse " primer 5 '- accaatgatctcatccgtttgtgtttcaaagaagacgta-3’(SEQ ID NO：6), and PCR2 reactions are used for " prominent - the tacgtcttctttgaaacacaaacggatgagatcattggt-3 ' of forward primer 5 ' (the SEQ ID NO of change "：7) given birth to ox Long hormone polyadenylation signal reverse primer (BGHR) 5 '-tagaaggcacagtcgagg-3 ' (SEQ IDNO：8).With GeneAmp PCR systems 9700 (Applied Biosystems) carry out the reactions of PCR1 and 2：94 DEG C 5 minutes, then carry out 30 times 94 DEG C 1 minute, 40 DEG C of 1 minute and 72 DEG C of circulations of 2 minutes.PCR1 and PCR2 reaction product is cut from Ago-Gel Under, then illustrate to purify according to manufacturer with Geneclean II kits (Anachem Ltd, Luton, LU2 OEB, UK).It is pure PCR1 the and PCR2 products of change are used to carry out PCR3 to build the complete TSHR sequences containing mutation.The PCR3 reactions include 200ng PCR1 products and 200ng PCR2 products.PCR3 carry out 7 94 DEG C 1.5 minutes, 65 DEG C 1.5 minutes and 72 DEG C 1.5 points The circulation of clock.Then temperature is increased to 94 DEG C up to 2 minutes again, then adds T7 primers and BGHR primers, then carry out 30 94 DEG C 1 minute, 52 DEG C of 1 minute and 72 DEG C of circulations of 2 minutes.With BamHI and XhoI restriction sites by the PCR3 products (TSHR E358T/E360T) is cloned into pcDNA 5.1/FRT carriers (Invitrogen), and with Sanger Coulson methods Confirm presence (the DNA sequencing with of Sanger F, Nicklen S, Coulson AR 1977 of mutation chainterminating inhibitors.Proceedings of the National Academy of Sciencesof the USA 74：5463-5467).

Then by the TSHR E358T/E360T constructs be used as template DNA use with above for TSHR E358T/ Method same E360T introduces the 3rd mutation (R312E)." reverse " primer 5 ' that PCR1 reactions are mutated using being directed to- gcattcacagatttttcctggcgcaagctctgca-3’(SEQ ID NO：9) and PCR2 reaction using for mutation " just To " 5 '-tgcagagcttgcgccaggaaaaatctgtgaatgc-3 ' (SEQ ID NO of primer：10).By the PCR1 of purifying and PCR2 products carry out PCR3 reactions as described above, with BamHI and XhoI restriction sites by the PCR3 products (TSHRE358T/ E360T/R312E) it is cloned into pcDNA 5.1/FRT carriers (Invitrogen), and is sequenced really with Sanger Coulson methods Recognize presence (Sanger F, Nicklen S, the Coulson AR1977 DNA sequencing with chain of mutation terminating inhibitors.Proceedings ofthe National Academy of Sciences of the USA 74：5463-5467).

Then the TSHR E358T/E360T/R312E (including three mutation) are used as the above-mentioned PCR side of template DNA Method lacks amino acid 313-353." reverse " primer 5 ' lacked using being directed to of PCR1 reactions- catccgtttgtgtttcaaagaagacttcctggcgcaagctctgcatactg-3’(SEQ ID NO：11), and PCR2 is anti- - the cagtatgcagagcttgcgccaggaagtcttctttgaaacacaaacgga of " forward direction " primer 5 ' for missing should be used tg-3’(SEQE ID NO：12).PCR1 the and PCR2 products of purifying are subjected to PCR3 reactions as described above, with BamHI and XhoI Restriction site will encode the C-del TSHR product clonings of the TSHR amino acid/11s -764 of residue 313-353 missings to pcDNA In 5.1/FRT carriers (Invitrogen), and with Sanger Coulson methods (Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chainterminating inhibitors.Proceedings of the National Academy of Sciencesof the USA 74：5463-5467) sequencing confirms missing, then as template With T7 promoter primers and 5 '-gctactcgagctagtggtggtggtggtggtggtcttcacacgggttgaactcatcg ga cttg-3’(SEQ ID NO：13) expanded, the result is that the C-terminal after TSHR amino acid 410 adds 6 histidines Label, a terminator codon and XhoI restriction sites.PCR reaction carry out 25 times 95 DEG C 1 minute, 50 DEG C 1 minute and 72 DEG C of circulations of 1 minute, the C-del TSHR are then cloned into pFastbacl with BamHI and XhoI restriction sites In (Invitrogen, UK), and with the SangerCoulson methods (DNA of Sanger F, Nicklen S, Coulson AR 1977 sequencing withchain terminating inhibitors.Proceedings of the National Academy ofSciences of the USA 74：5463-5467) sequencing confirms DNA sequence dna.TSHR260 restructuring is made Bacmid dna and the presence that restructuring rod granule is confirmed with PCR.Restructuring rod granule DNA is transfected into Sf-9 insect cells, as described above for Virus stocks are prepared described in TSHR260 and are titrated.

Purify M22 IgG and Fab preparation

Use MabSelect^TMAlbumin A affinity chromatography (GE Healthcare, UK) is from hetero hybridoma culture supernatants system M22 IgG, such as Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Kiddie A, Brereton K, Premawardhana LDKE, Chirgadze DY,Miguel R, Blundell TL, Furmaniak J, Rees Smith B2004 Characteristics of a human monoclonal autoantibody to thethyrotropin receptor：sequence structure and function.Thyroid 14：Described in 560-570.It is pure with mercury papain (Sigma, UK) processing with the ratio of enzyme/albumen 1: 50 The IgG of change, then passes through MabSelect^TMPost removes any complete IgG or Fc from Fab preparations, such as Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Kiddie A, Brereton K, Premawardhana LDKE, Chirgadze DY,Miguel R3Blundell TL, Furmaniak J, Rees Smith B 2004 Characteristics of a humanmonoclonal autoantibody to the thyrotropin receptor：sequence structureand function.Thyroid 14：Described in 560-570.In reductive condition It is lower to analyze M22 Fab with sds polyacrylamide gel electrophoresis (SDS-PAGE) to determine purity.With expression TSHR Chinese hamster Ovary (CHO) cell detects M22 Fab biological activity by cyclic adenosine monophosphate irritant test.In addition, also analyze M22 Fab Suppress¹²⁵I-TSH or¹²⁵Ability (Sanders J, Oda Y, the Roberts S3 Kiddie that I-M22 is combined with TSHR coating pipes A3 Richards T3Bolton J3 McGrath V3 Walters S3 Jaskolski D3 Furmaniak J3 ReesSmith B 1999 The interaction of TSH receptor autoantibodies with125I- labelled TSH receptor.Journal of Clinical Endocrinology andMetabolism 84： 3797-3802)。

The preparation of TSHR 260-M22 compounds

By High Five^TMInsect cell (BTI-TN-5B1-4, Invitrogen, UK) is placed in added with 0.1mmol/L In the EXCell culture mediums of KI and 7 μ g/mL gentamicins, under 22 DEG C, 60rpm ventilation stirring condition, in 500mL rotation Cultivated in flask (Techne).Cell-seeding-density in each flask is 0.5x10⁶Cell/mL, and cultivate 24 at 22 DEG C Hour, then infected with baculoviral original seed, infection multiplicity (MOI) is 0.0006pfu/ cells.Continue to cultivate cell culture, And add the M22Fab, final concentration of 2 μ g/mL purified within 96 hours after infection.Centrifuge 10 by 500g within 120 hours after infection Minute culture supernatants of the harvest containing TSHR260-M22 Fab compounds.A piece of Complete is added per 200mL supernatants Protease inhibitors (Roche), -70 DEG C are then stored at until purifying.

Carry out a series of independent measurings in the absence of M22 IgG, exist 8 μ g/mL M22IgG and exist 12 μ g/ In the case of mL M22 IgG, TSHR260 produced in High Five insect cell cultures during i.e. after infection the 3rd, 4, The stability of 5 and 6 days.M22 IgG are added in High Five cell culture supernatants on the day of infection.To culture supernatant Each sample carry out western blot analysis, so as to determine the TSHR260 of expression integrality (Fig. 1 a).With 1 μ g/mL concentration can The mouse monoclonal antibody that TSHR epitopes (TSHR MAb 18C5) between amino acid 246-260 react carries out albumen Trace (Jeffreys J, Depraetere H, Sanders J, Oda Y, Evans M, Kiddie A, Richards T, Furmaniak J, Rees Smith B 2002Characterization of the thyrotropin binding pocket.Thyroid 12：1051-1061).

Specifically, Fig. 1 a are represented in the case where being not present or M22 IgG being present the 3rd, 4,5 and 6 day after infection (respectively For swimming lane 1-4) TSHR260 that detects in High Five cell culture supernatants.Illustration 1=is in the feelings in the absence of M22 IgG From the expression TSHR 260 cell culture supernatant sample that obtains of High Five cells, (swimming lane 1-4 represents infection respectively under condition The the 3rd, 4,5 and 6 day afterwards, swimming lane 5 was the HighFive cultures obtained from the cell for not expressing TSHR 260 as negative control Supernatant).Illustration 2=obtains in the case where 8 μ g/mL M22 IgG be present from expression TSHR 260 High Five cells Cell culture supernatant sample (swimming lane 1-4 represent respectively infection after the 3rd, 4,5 and 6 day, swimming lane 5 is from as negative control Do not express TSHR260 cell obtain High Five culture supernatants).Illustration 3=has 12 μ g/mL M22IgG's In the case of from the expression TSHR 260 cell culture supernatant sample that obtains of High Five cells, (swimming lane 1-4 represents sense respectively The the 3rd, 4,5 and 6 day after dye, swimming lane 5 is the High Five trainings obtained from the cell for not expressing TSHR 260 as negative control Support supernatant).

As shown in Figure 1a, cultivated under conditions of in the absence of M22 IgG, represent TSHR 260 molecular weight as 34kDa's The intensity of band increases to maximum on the 5th day after infection.However, the 6th day (illustration 1 after infection；Swimming lane 4) most of TSHR 260 Product degradation is the albumen that molecular weight is 29kDa or the condensation zone for forming 41kDa.When in culture medium added with 8 μ g/mL M22 During IgG, similar pattern can be observed, although at the 6th day except the thing of degraded or polymerization compared with the experiment in the absence of M22 IgG More complete TSHR 260 (Fig. 1 a illustrations 2) outside matter also be present.When the M22 IgG concentration in culture medium increases to 12 μ g/ It is similar to the 5th day in the amount for the complete TSHR 260 that the 6th day detects during mL.

These results indicate that TSHR 260 is protected in the medium by forming compound with M22 IgG.Pass through other High when experiment further study M22 Fab in the presence of (adding culture medium, final concentration of 2 μ g/ml on the 4th day after infection) The stability of TSHR 277 and C-del the TSHR products of Five insect cell expressions.The 5th day from by with corresponding after infection The High Five cells of the virus infection of TSHR constructs collect culture supernatant, and with Streamline HST matrix Chromatographic fraction purifies (as described below).Western blot analysis is carried out to each culture supernatant sample, so as to determine the TSHR of expression The presence of product and molecular weight (Fig. 1 b and c).With 1 μ g/mL concentration can be with the TSHR epitopes between amino acid 246-260 Mouse monoclonal antibody that (TSHR MAb 18C5) reacts carry out western blot (Jeffreys J, Depraetere H, Sanders J, Oda Y, Evans M, Kiddie A, Richards T, Furmaniak J, Rees Smith B 2002Characterization of the thyrotropin binding pocket.Thyroid 12：1051-1061).

Specifically, Fig. 1 b are shown before purification and the culture supernatant for being present in collection with Streamline post parts after purification TSHR 277, the C-del TSHR and TSHR 260 of liquid.The cell training of swimming lane 1=expression TSHR 277 High Five cells Support supernatant samples, the partially purified TSHR 277 of swimming lane 2=.Swimming lane 3=is present in the C-del of cell culture supernatant TSHR, the identical material after swimming lane 4=partial purifications.Swimming lane 5 and 6 is respectively the TSHR 260 before and after partial purification.In swimming lane 2 With 4 in TSHR MAb 18C5 and M22 Fab (molecular weight 46kDa) some non-specific bindings can be observed.

As shown in Figure 1 b, the High Five cells cultivated under the conditions of existing for M22 Fab express molecular weight 34kDa TSHR 277, however, TSHR 277 decrease in molecular weight is to 30kDa (Fig. 1 b swimming lanes 1 and 2) after partial purification. Under the conditions of M22 Fab are existing, the C-del of High Five cells expression is detected in cell culture supernatant sample TSHR, 46kDa protein bands are shown as, it is degraded to 30kDa albumen (Fig. 1 b swimming lanes 3 and 4) after partial purification.It is herein TSHR 260 in row experiment is detected as 30-32kDa albumen (Fig. 1 b swimming lanes 5 and 6) afterwards before purification.

This experiment display, there is TSHR277 the and C-del TSHR productions of expected molecular weight (being respectively 34kDa and 46kDa) Thing is expressed in High Five cells, but the size that both albumen are all degraded after partial purification to TSHR260 albumen is (big About 30kDa).

Another example that C-del TSHR degrade in partial purification is as illustrated in figure 1 c.In the M22Fab that 2 μ g/mL be present When, C-del TSHR are expressed in High Five cells.

Specifically, Fig. 1 c show in High Five cell culture supernatants and purified in StreamlineHST matrix Different phase C-del TSHR.TSHR 260 in swimming lane 1=High Five cell culture supernatants；Swimming lane 2=High C-delTSHR in Five cell culture supernatants；Swimming lane 3=expression C-del TSHR High Five cells and supernatants Liquid, it is diluted and adjusted to can be loaded to Streamline HST posts；The material that swimming lane 4=passes through Streamline HST posts Material；The Streamline HST post fractions containing partial purification C-del TSHR of swimming lane 5-8=elutions.

As illustrated in figure 1 c, C- of the High Five cells cultivated under the conditions of M22Fab is existing by molecular weight for 46kDa Del TSHR are expressed into culture supernatant (swimming lane 2).Also detected in the loading material to StreamlineHST posts complete C-del TSHR (swimming lane 3), do not detect C-del TSHR (swimming lane 4) in post efflux.However, the C- eluted from post It is about 30kDa (swimming lane 5-8) that del TSHR, which degrade to molecular weight, suitable with the molecular weight of the TSHR260 shown in swimming lane 1.

This serial experiment illustrates, in the presence of M22Fab, has the TSHR polypeptide chains of heterogeneous expectations length by used Construct is expressed in High Five cells.However, even in purifying early stage, TSHR 277 and C-del TSHR products also can It is degraded to the size of the products of TSHR 260.Evidence show TSHR 260 can degrade and (also see below) in purifying.Therefore, most may be used Can be M22Fab combination be related to most of polypeptide chains of TSHR 260 and protect it from protein hydrolysis.TSHR amino acid C-terminal of the sequence since residue 260 is less likely the stable bond for participating in M22Fab, therefore amino acid 260 and 277 or 260 And the TSHR sequences between 410 are by protein degradation.

TSHR260-M22Fab compounds have surprising stability.This with it is former it is unstable, under Production conditions it is fast The TSHR polypeptide formulations of speed denaturation are opposite.The TSHR260-M22Fab compounds with endoglycosidase F3 by removing glycosyl After changing front and rear two-wheeled affinity purification, analyzed with HPLC gel filtrations and SDS-PAGE electrophoresis, as shown in figs. 2 a-c.

Specifically, Fig. 2 a are shown：The TSHR 260-M22 Fab compounds purified before deglycosylation --- pass through gel Filtering HPLC analysis (TSK-GEK G3000SW, is carried out in 150mmol/LNaCl, 10mmol/L Tris pH 7.0；Level Part volume is 0.5mL).Peak 1=TSHR260-M22 Fab compounds.Peak 2=only has M22Fab, with the analysis knot individually carried out Fruit overlaps.Fig. 2 b are shown in the TSHR 260-M22 Fab compounds purified after deglycosylation, in cation exchange HPLC Separation and concentration；The material for being used to crystallize --- analyzed by gel filtration HPLC, as shown in Figure 2 a.Peak 1=TSHR 260-M22 Fab compounds, the free M22 Fab of peak 2=.Shown in Fig. 2 c and SDS-PAGE (12% is used under non reducing conditions Acrylamide gel) analysis to the TSHR260-M22 Fab of purifying, and indicate the position of molecular weight marker.Mark M22 Fab and TSHR 260 position before and after deglycosylation.TSHR260-M22 Fab before swimming lane 1=deglycosylations；Swimming lane 2 After=deglycosylation and on cation exchange HPLC post before purification；Swimming lane 3=deglycosylations and in cation exchange HPLC post On after purification；Swimming lane 4=deglycosylations and on cation exchange HPLC post after purifying and final concentration.Swimming lane 1,2 and 4 is often swum The road about μ g of loading 10 TSHR260-M22 Fab, the about μ g of loading 5 of swimming lane 3.

As shown in Figure 2 a, the analysis result of TSHR260-M22 Fab compounds is substantially single spike (peak 1), only (5%) free M22 Fab are present in sample on a small quantity.Deglycosylation and on cation exchange HPLC post separate after, such as Judged by gel filtration HPLC, TSHR260-M22 compounds globality is complete (Fig. 2 b peaks 1), in the end system for crystallization A small amount of (7%) free M22 Fab (Fig. 2 b peaks 2) are only detected in agent.

This compound is decomposed into ratio by analyses of the SDS-PAGE to the TSHR260-M22 Fab of purifying under non reducing conditions Two roughly equal component (TSHR260 and M22 Fab of example；Fig. 2 c swimming lanes 1).When being calculated according to SDS-PAGE, glycosylation M22 Fab molecular weight is about 56kDa and TSHR260 molecular weight is about 40kDa.After all deglycosylations sample ( Before being separated by cation exchange HPLC with endoglycosidase F3, after cation exchange HPLC and after concentration；Fig. 2 c, respectively swim Road 2,3 and 4) all show similar albumen band model.Topmost the minimum band of about 56kDa ratio represents not going for some remnants Glycosylated M22 Fab.Molecular weight about 54kDa protein band represents deglycosylated M22 Fab, and molecular weight about 37kDa Protein band represents deglycosylated TSHR260.

The N-terminal amino acid analysis (such as Fig. 2 c, shown in swimming lane 1) of molecular weight about 40kDa protein band shows following sequence Row：Met-Gly-X-Ser-Ser-Pro.Wherein X may be Cys.Sequence between this corresponding mankind's TSHR amino acid 22-27 is Met-Gly-Cys-Ser-Ser-Pro is simultaneously consistent with originating in the expressed sequence of residue 22 (residue 1-21 is signal peptide) (Cloning of Misrahi M, Loosfelt H, Atger M, Sar S, Guiochon-Mantel A, Milgrom E 1990, sequencing and expression of human TSH receptor.Biochemical andBiophysical Research Communications 166：394-403).Therefore, we are determined by N-terminal amino acid sequencing and purified Compound in TSHR260 property.

The purifying of TSHR260-M22 Fab compounds

With 500mmol/L NaH₂Culture supernatant (14.4 and 17.4L two batches) regulation is arrived pH 6.2 and loading by PO4 To the 75mL treamline Direct HST bases in Streamline25 expanded bed chromatographies system (GE Healthcare, UK) In matter.Another batch of culture supernatant (11.9L) is handled in the same fashion in independent experiment.With 50mmol/L sodium phosphates (pH 6.0), 50mmol/L NaCl are washed, then are washed with 100mmol/L NaCl, 50mmol/L Tris-HCl (pH 6.5), are used in combination 100mmol/L NaCl, 50mmol/L Tris-HCl (pH 8.0) are eluted.With 1 μ g/mL concentration can be with amino acid 246-260 Between the mouse monoclonal antibody that reacts of TSHR epitopes (TSHR MAb 18C5) carry out western blot to confirm to elute level Part in TSHR260-M22 compounds presence (Jeffreys J, Depraetere H, Sanders J, Oda Y, Evans M, The Characterization of of Kiddie A, Richards T, Furmaniak J, Rees Smith B 2002 thethyrotropin binding pocket.Thyroid 12：1051-1061).

Pass through affine color with the antibody TSHRMAb 14C4 for being coupled to CNBr activated sepharoses -4B (Sigma, UK) again Spectrum is purified (Jeffreys J, Depraetere H, Sanders J, Oda Y, Evans to TSHR260-M22 compounds The Characterization of the of M, Kiddie A, Richards T, Furmaniak J, Rees Smith B 2002 thyrotropinbinding pocket.Thyroid 12：1051-1061), the ammonia of the antibody and TSHR ectodomains Comformational epitope between base acid 22-261 combines.In short, the compound is loaded in 4mL affinity columns, 100mmol/L is used NaCl, 50mmol/L Tris-HCl (pH 8.0) are washed, with 100mmol/L NaCl, 100mmol/L citrates (pH 4.0) Elution, is collected into isometric neutralization buffer (0.5mol/L Tris-HCl, pH 8.0), then carries out dialysis 50mmol/L NaCl、10mmol/L Tris-HCl(pH 8.0)。

Then the compound through dialysis is further purified with Nickel affinity chromatographies.The compound is loaded to Ni- In NTA agarose columns (Qiagen, UK), with lavation buffer solution (50mmol/LNaCl, 10mmol/L Tris-HCl pH 8.0) Washing is again with the lavation buffer solution eluting cmp added with 20mmol/L imidazoles.The compound is carried out dialysis into 50mmol/L In NaCl, 10mmol/LTris-HCl (pH 8.0) and for carrying out deglycosylation reaction.The concentration of the compound according to 280nm trap calculates, and its basis is TSHR260-M22 Fab of 1 absorbance units equivalent to 0.69mg/mL.

The deglycosylation of TSHR 260-M22 compounds and final purification

It will be cultivated with endoglycosidase F3 (Sigma, UK) in pH 4.5 50mmol/L sodium-acetate buffers from 31.8L The compound desaccharification for 16mg (or the 7.5mg obtained in another experiment from the 11.9L culture supernatants) purifying that supernatant obtains The ratio of base, enzyme and compound is 152mU/mg compounds, reacts and is carried out 5 days at 20 DEG C.The deglycosylation is reacted Thing is loaded to cation exchange HPLC Bioassist S posts (TOPOH).In short, with 200mmol/LTris-HCl (pH 6.8) with reactant described in 1: 1 dilution proportion, filtered by 0.22 μm of filter, then upper prop.Then 20mmol/L is used NaCl (pH 6.5) washs the post, then elutes the compound with pH gradient liquid (pH 6.5-pH 9.0).Then use 5mg (or 2.4mg in another experiment) compounds purified are concentrated to by Microcon YM-10 inspissators (Millipore, UK) 32.8mg/mL (or 32mg/mL), determined with HPLC tsk gel G3000SW posts (TOSOH) by gel-filtration analysis described The integrality of compound simultaneously determines purity by SDS-PAGE analyses.This material, which is used for crystallization screening, to be tested or with aliquot Form is stored in -20 DEG C.

TSHR 260 protein sequencing

The TSHR260-M22 Fab compounds (30 μ g) of purifying are run into glue (in non reducing conditions on 12%SDS-PAGE Under), then trace is in 10mmol/L 3- Cyclohexylamino -1- propane sulfonic acid (CAPS) (pH 11) and 10% methanol Immobilon P^SQOn transfer membrane (Millipore, Watford, UK).By the film coomassie brilliant blue staining, representative is cut TSHR260 band simultaneously analyzes N-terminal amino acid sequence (Alta Biosciences, Birmingham, UK).

The amino acid mutation of M22 heavy chains (HC) or light chain (LC)

" C " end is cloned into the carrier derived from pUC18 with 6 histidine-tagged M22HC and LC sequences, such as Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Kiddie A, Brereton K, Premawardhana LDKE, Chirgadze DY, Nunez Miguel R, Blundell TL, Furmaniak J, Rees Smith B 2004Characteristics of a human monoclonal autoantibody to the thyrotropinreceptor：sequence structure and function.Thyroid 14：Described in 560-570.

Specific " forward direction " and " reverse " PCR primer is devised for each mutation, to change HC or LC nucleotides volume Code sequence encodes correct amino acid mutation.The primer is prepared by SigmaGenosys (Cambridge, UK).Carry out two Individual independent PCR reactions (PCR1 and PCR2).Reacted for LC PCR1, using M13 reverse sequencing primers and for mutation " reverse " primer；And reacted for PCR2, use " forward direction " primer for mutation and -20M13 " forward direction " sequencing primer.For HC PCR1 reactions, use M13 " reverse " sequencing primers and " forward direction " primer for mutation；And reacted for PCR2, use For " forward direction " primer and -20M13 " forward direction " sequencing primer of mutation.Use GeneAmp PCR System 9700 (AppliedBiosystems, UK) carries out PCR1 and PCR2 reactions, and first then 94 DEG C carry out 94 DEG C 1 minute, 42 DEG C 1 in 5 minutes Minute and 72 DEG C of 30 circulations of 2 minutes.PCR1 and PCR2 products are cut from Ago-Gel and are tried with Geneclean II Agent box (Anachem Ltd, UK) is purified according to manufacturer's explanation.Then PCR1 the and PCR2 products of purifying are used to carry out PCR3 with build containing mutation complete HC or LC sequences.The PCR3 reactants include 200ng PCR1 products and 200ng PCR2 products.PCR3 carry out 7 94 DEG C 1.5 minutes, 65 DEG C of 1.5 minutes and 72 DEG C of circulations of 1.5 minutes.Then by temperature again Degree is increased to 94 DEG C up to 2 minutes, addition -20M13 " forward direction " sequencing primers and M13 " reverse " sequencing primer, then carries out 30 94 DEG C 1 minute, 42 DEG C of 1 minute and 72 DEG C of circulations of 2 minutes.

Wild type or the M22 HC of mutation are cloned into Xhol and Spel restriction sites, and by wild type or mutation M22 LC are cloned into Immunozap H/L carriers (Stratagene Europe；Amsterdam, Netherlands) SacI In Xbal restriction sites, and the Sanger-Coulson methods sequencing of used this area confirms the presence of mutation.

DNA containing M22HC and LC sequences is transformed into HB2151 cells (Amersham Bio sciences) And preculture；LB ampicillin mediums (the pancreas egg that a colony for being converted HB2151 is contained into 1% glucose in 10mL White peptone 10g/L, yeast extract 5g/l, NaCl 10g/l, the μ g/mL of ampicillin 100) at 30 DEG C shake overnight incubations. Then pre-culture is diluted into (5mL is diluted in 500mLLB ampicillin mediums) and cultivated at 30 DEG C, until 600nm Absorption value reaches 1.2.Then 1.8mol/L sucrose is added to final concentration of 0.3mol/L, and culture is cultivated directly at 30 DEG C 1.2 are returned to 600nm absorption values.It is subsequently to added into isopropanol-β-D thiogalactosides (IPTG) extremely final concentration of 1mmol/L simultaneously Culture is cultivated 24 hours in 23 DEG C of continuous shake.Then culture is centrifuged 30 minutes at 4 DEG C with 9000rpm, added 1mmol/L phenylmethylsulfonyl fluorides (PMSF) and every 100ml supernatants add 1 Complete protease inhibitors (Roche), and Supernatant is placed in -70 DEG C of storages before purification.Western blot is carried out with anti-human igg (Fab is special) antibody (Sigma, UK) It is analyzed to identify the expression of recombinant Fab.Western blot analysis can detect 10ng wild type restructuring M22.

Recombinate wild type and be mutated M22 Fab purifying

The supernatant containing restructuring M22 Fab (is purified with 4 every time with 500mmol/L sodium dihydrogen phosphate (pH 4.0) Rise) adjust to pH 6.0 and be loaded on 75mLStreamline Direct HST posts (GE Healthcare, UK).With 10mmol/L Tris-HCl (pH 6.8), 0.1mol/LNaCl wash the post until 280nm absorption values reach less than 0.1, then With 0.3mol/L NaCl and 10mmol/L Tris-HCl (pH 8.3) elution M22 Fab.The material of elution is loaded to Ni- In NTA agarose columns (Qiagen, UK), with 0.3mol/L NaCl and the 10mmol/L Tris-HCl containing 40mmol/L imidazoles (pH 8.3) is washed, then elutes M22 Fab with 120mmol/L imidazoles.

The Fab of elution purity ＞ 95% is measured by SDS-PAGE, and the dense of Fab is calculated according to 280nm absorption value Degree, its basis is 1 Fab of the absorbance units equivalent to 0.7mg/mL.

The amino acid mutation of mankind's TSHR sequences

For introducing the method for specific mutation in TSHR sequences as described in WO2006/016121 A.

In short, BamHI and Xhol restriction sites are utilized by TSHR full length nucleotide sequences gram according to Standard cloning methods It is grand to arrive in pcDNA5.1/FRT carriers (Invitrogen).Specific " forward direction " and " reverse " PCR is devised for each mutation Primer, nucleotide coding sequence is changed to encode correct amino acid mutation.All primers are all by Sigma Genosys It is prepared by (Cambridge, UK).Carry out two independent PCR reactions (PCR1 and PCR2).PCR1 reactions are using T7 and for mutation " reverse " primer, and PCR2 reaction using for mutation " forward direction " primer and bovine growth hormone polyadenylation signal reversely draw Thing (BGHR primers).The reactions of PCR1 and 2 are carried out with GeneAmp PCR systems 9700 (Applied Biosystems)：94℃ 5 Minute, then carry out 30 times 94 DEG C 1 minute, 40 DEG C of 1 minute and 72 DEG C of circulations of 2 minutes.By PCR1 and 2 product from fine jade Cut on sepharose, then with Geneclean II kits (Anachem Ltd, Luton, LU2 OEB, UK) according to manufacturer Illustrate purifying.PCR1 the and PCR2 products of purifying are used to carry out PCR3 to build the complete TSHR sequences containing mutation.It is described PCR3 reactants include 200ng PCR1 products and 200ng PCR2 products.PCR3 carries out 7 94 DEG C 1.5 minutes, 65 DEG C 1.5 Minute and 72 DEG C of circulations of 1.5 minutes.Then temperature is increased to 94 DEG C up to 2 minutes, adds T7 primers and BGHR primers, then Carry out 30 94 DEG C 1 minute, 52 DEG C of 1 minute and 72 DEG C of circulations of 2 minutes.With BamHI/XhoI restriction sites by described in PCR3 product clonings are and true with the Sanger Coulson methods of this area into pcDNA 5.1/FRT carriers (Invitrogen) Recognize the presence of mutation.

The TSHR constructs of mutation are transfected into Chinese hamster ovary celI with FIp-In systems

Method therefor is as described in WO2006/016121A.In short, the FIp-In systems (Invitrogen) are used to incite somebody to action Wild type (Wt) and mutation T SHR cDNA transfections are to Chinese hamster ovary celI.Each FIp-In-CHO cells contain a FIp-In site, Therefore TSHR DNA can be inserted into the same position of genome in each experiment, and can only have a copy per cell. The FIp-In-CHO cells that one flask converges are used for being inoculated with 24 orifice plates, the inoculation 1x10 per hole⁵-1.5x10⁵Individual cell, Kong Zhonghan There are DMEM (Invitrogen) and 10% hyclone (FCS) (Invitrogen), without antibiotic, by the cell 37 DEG C, 5%CO₂Cultivated overnight with conditions of the humidity of ＞ 95%.Then with lipofectamine (Invitrogen) according to manufacturer Illustrate to transfect pcDNA5.1/FRT TSHRDNA and POG44DNA (Invitrogen) into FIp-In Chinese hamster ovary celIs.With containing 600 μ The culture medium selection cell of g/mL hygromycin (Invitrogen), is transfected by POG44 DNAs and pcDNA5.1/FRT TSHR Those cells TSHR can be inserted into FIp-In-CHO cellular genomes and show hygromycin resistance.

Analysis to the stimulation of cyclic adenosine monophosphate generation

WO2006/016121A is described in detail for detecting to by the Chinese hamster ovary celI middle ring of wild type or mutation T SHR transfections The stimulation of adenylate generation, to confirm the method for the function of the construct.

In short, Chinese hamster ovary celI is inoculated into 96 orifice plates into (per 12500-20000, hole cell), and containing 10% tire Cultivated 48 hours in the DMEM of cow's serum.Then DMEM is removed, add cyclic adenosine monophosphate analysis buffer (be free of NaCl, but Contain 1g/L glucose, 20mmol/L HEPES, 222mmol/L sucrose, 15g/L bovine serum albumin(BSA)s (BSA) and 0.5mmol/l 3-isobutyl-1-methylxanthine Hank ' s buffering salting liquid, pH 7.4) in dilution pig TSH (RSR Ltd；0.01- 3ng/mL) and wild type or mutation M22 Fab (0.1-10ng/mL), then have 5% CO in 37 DEG C, air₂Atmosphere under Cultivate 1 hour.After removing test solution, cell lysis and with Amersham Biosciences Biotrak Dot Enzyme Immunoassays Cyclic AMP concentrations in system measurement lysate.

It is dissolved in the wild type of detergent and the preparation of mutation T SHR preparations

Method therefor is as described in WO2006/016121A.

Wild type or mutation T SHR FIp-In-CHO cells are expressed in 175cm²Flask in cultivate to converging, use DulbeccoShi PBS (not calcic and magnesium ion) (Invitrogen) wash cell, then scrape 10mL ice-cold buffers A In (50mmol/L NaCl, 10mmol/L Tris-HCl, pH 7.5), buffer solution contains 1mmol/L phenylmethylsulfonyl fluorides and every 200mL buffer solutions contain 1 Complete protease inhibitors (Roche Diagnostics).The cell at 4 DEG C with 1000xg, which is centrifuged, to be precipitated, then is homogenized by pellet resuspended in 1mL buffer As and on ice with glass homogenizer.Cell Film is centrifuged 30 minutes with 12000xg at 4 DEG C and precipitated, then is resuspended to added with 0.5g/L sodium azide and 2.75g/L6mL In the buffer A of iodoacetamide, and precipitate again as described above.Then by film sediment be resuspended to 1mL it is ice-cold contain 1% In the buffer A of Triton X-100 and 0.5g/L sodium azide and it is homogenized.The dissolved TSHR preparations at 4 DEG C with 9000xg is centrifuged 2 hours, and supernatant is stored in into -70 DEG C.

It is right¹²⁵I-M22IgG or¹²⁵The suppression that I-TSH is combined with TSHR

Carried out with the test tube for being coated with wild type TSHR¹²⁵I mark M22IgG or¹²⁵The TSH of I marks, which is combined, to be suppressed to try Test, such as Sanders J, Oda Y, Roberts S, Kiddie A, Richards T, Bolton J, McGrath V, Walters The The interaction of TSH receptor of S, Jaskolski D, Furmaniak J, Rees Smith B 1999 autoantibodies with125I-labeled TSH receptor.Journal of Clinical Endocrinology andMetabolism 84：(reagent is purchased from RSR Ltd) described in 3797-3802.Each experiment includes One calibration curve (1-100ng/mL) prepared with M22 Fab, the M22 Fab are with from M22 Hybridoma Cell Culture things Prepared by the IgG of supernatant purifying.

In this test, by the wild type of 100 μ L purifying or the Fab of mutation (in experiment buffer solution (50 mmol/L NaCl, 10mmol/L Tris-HCl pH 7.8,0.1%Triton X-100) in be diluted to 0.001-100 μ g/mL) be placed in Cultivation 2 hours is gently shaken in the coated test tubes of TSHR at room temperature.After extracting liquid out, test tube is washed with 1mL experiment buffer solutions Twice, 100 μ L are then added¹²⁵I-M22 IgG (50,000cpm) or¹²⁵I-TSH (80,000cpm) simultaneously shakes training at room temperature Support 1 hour.Then test tube is washed twice with 1mL experiment buffer solutions, sucking liquid is simultaneously counted with gamma counter.To M22 IgG or The suppression that TSH is combined calculates according to following formula：

Crystallization and the collection of diffraction data

The deglycosylation TSHR-M22 Fab compounds that concentration is 32.8mg/mL are used for steam and spread hanging drop crystallization experiment. Went out after about two weeks in Wizard Crystal Screen I, condition #46 (Emerald BioStructures, Inc.) Existing small crystals cluster.Then by crystallization solution in 8%PEG8000,0.1mol/L MES pH 6.0,0.25mol/L zinc acetates it is excellent Change, this can make crystallization bigger but still can cluster.The use of macroscopic view/microcosmic seeded crystallization technique to obtain the trial of monocrystal is unsuccessful 's.It is 0.02x0.02x0.05mm by size³Monocrystal from cluster manually separation and containing 26% ethylene glycol (as freezing Protective agent) liquid nitrogen in quickly cool down.

With " self-control " copper wheel anode radioactive source, (generator RU-H3R, Rigaku-MSC Ltd., is total to equipped with Max-Flux Focus on multilayer film eyeglass, Osmic Inc.) carry out X ray diffracting data collection experiment in 100K.With Raxis IV++ image plates Detector (Rigaku-MCS Ltd.) records diffraction data.Collected once swinging step (collecting 129 degree altogether) with monocrystal Original diffraction data, then handle suit with HKL diffraction datas and index, integrate, measure and reduce these data (Otwinowski The Processing of X-ray diffraction datacollected in oscillation of Z, Minor W 1997 mode.Methods in Enzymology：MacromolecularCrystallography, part A 276：307-326). The crystal belongs to orthogonal I2₁2₁2₁Space group, it has TSHR-M22 Fab compound (54% solvents in asymmetry unit Content) and it is diffracted into 3.1Bragg ' s spacing.(refinement) statistics of refining is as shown in table 1.

Prepare TSHR260 and M22 Fab compound again by description, be concentrated into 32mg/mL and carry out crystallization trial, according to Crystal is obtained with First Series experiment identical crystallization condition.With synchrotron radioactive source (work station PXI 4.2, Council For the Central Laboratory of theResearch Councils, Daresbury, UK) carry out X-ray diffraction Data Collection.Data are collected from monocrystal and diffraction resolution is improved to 2.55 in 100KBragg ' s spacing.Then with new 2.55 obtainedResolution data will be with 3.1Resolution ratio obtain initial configuration refine again, to the R factors (R- Factor) it is 18.1% (R_free=24.5%).Statistics of refining is as shown in table 1.

As a result

Structure determination and refine

The deglycosylated TSHR260-M22 Fab compounds in part successfully crystallize but only generate polycrystal cluster, the crystal Cluster can not be developed further to obtain monocrystal.Monocrystal suitable for X-ray diffraction analysis can be obtained by division crystal cluster by hand .

The structure is replaced by molecule and parsed.M22 Fab (Sanders J, Jeffreys J, DepraetereH, Evans M, Richards T, Kiddie A, Brereton K, Premawardhana LDKE, Chirgadze DY, Nunez Miguel R, Blundell TL, Furmaniak J, Rees Smith B2004 Characteristics of a human monoclonal autoantibody to thethyrotropin receptor：sequence structure and function.Thyroid 14：560-570) and FSHR (Fan QR, Hendrickson WA 2005Structure of humanfollicle-stimulating hormone in complex with its receptor.Nature 433： 269-277) crystal structure is used as molecule and replaces search model；Calculate in the CCP4 program groups (The of Bailey S 1994 CCP4 suite-programs for protein crystallography.Acta Crystallography Section D 50：AmoRe (Navaza J 1994Amore-an automated package for molecular 760-763) replacement.ActaCrystallography Section D 50：Completed on 157-163).M22Fab search probes enter One step is divided into two：One comprises only variable region, and another is containing only constant region.Successfully obtained in asymmetry unit TSHR and The position of M22 Fab variable regions, it result in the 48.2% R factors.But constant region is not parsed, therefore use is Electron-density map manual placement these constant regions that one wheel calculates after refining.Before refining, obtained model has 43.5% R The factor and 45.5% R_free.8 wheel crystallography are carried out altogether to refine and artificial implants.Atomic crystal is refined using CNS (Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nigles M, Pannu NS, ReadRJ, Rice LM, Simonson T, Warren GL 1998 Crystallography and NMRsystem：a new software suite for macromolecular structure determination.Acta Crystallography Section D 54：905-921), and in the rear stage Use REFMAC (the Refinement ofmacromolecular of Murshudov GN, Vagin AA, Dodson EJ 1997 structures by the maximum-likelihood method.ActaCrystallography Section D 53： 240-255) bag is completed.The simulated annealing method used in CNS is refined for preceding two-wheeled, but by Powell in step below Minimum method substitutes.The 2Fo-Fc weighted using sigmaA, Fo-Fc and annealing omit figure in Coot (Emsley P, Cowtan K2004 Coot：model-building tools for assessing the accuracy of crystalstructures.Nature 355：Artificial implants are carried out on 472-475).Zn²⁺Ion, 2-Acetamido-2-deoxy-D-glucose Residue and hydrone be served only for the later stage refine/reconstruction procedures on.Composite structure model is 3.1Refined in resolution ratio to R The factor is 20.7% (R_free=28.3%).Then use 2.55The data that resolution ratio newly obtains are refined 3.1 again Initial configuration to the R factors that resolution ratio obtains are 18.1% (R_free=24.5%) (table 1).

Final structure is by M22LC (residue 1-208), HC (residue 1-127 and 134-213), TSHR (residue 30-257), 6 Individual 2-Acetamido-2-deoxy-D-glucose residue, 5 zinc ions and 289 hydrone compositions.In all N linked glycosylation sites of TSRH Observe continuous electronic density in (N77, N99, Nl13, Nl77 and Nl98) and M22 a site (LC N26).Due to unordered, M22 HC128-133 cyclic residue and some terminal residues (M22 Fab LC 209-212, M22Fab HC 214-220, TSHR residues 22-29,258-260 and C-terminal 6- histidines) there is no electron density.TSHR E35 pendant atom lacks clear Electron density, therefore the residue is modeled as alanine.

One example of electron-density map is referring to Fig. 3.Specifically, Fig. 3 shows TSHR (generally towards at the top of the figure) and M22 Part interface between Fab heavy chains (generally towards the figure bottom).The figure sketches out profile in 1.2 σ levels, all displays it is residual Base is all labeled.

TSHR structures

TSHR is in slight bending tubulose, there is relative convex-concave surface, one 10 chain beta sheet for being located at concave surface of band.The interior table of pipe Face is booked hydrophobic residue.Homologue closest to TSHR is FSHR, and it shares 40.9% sequence identity (Misrahi with TSHR The Cloning of M, Loosfelt H, Atger M, Sar S, Guiochon-Mantel A, Milgrom E 1990, sequencing and expressionof human TSH receptor.Bioc hemical and Biophysical ResearchCommunicatious 166：394-403 (Figure 10 b) and Fan QR, Hendrickson WA 2005Structure of human follicle-stimulating hormone in complex with its receptor.Nature 433：269-277)；C between both structures_αRoot-mean-square-deviation (rmsd) on core atom is 1.1There are 10 β chains in the concave surface of repetitive structures of the mankind TSHR rich in leucine, and form 9 in a parallel β-pleated sheet Repeat.Residue quantity of the every chain since N- ends is：4、5、5、5、5、7、5、6、3、3.That is in first repetition chain in addition β chains before then form beta hairpin structure.There are 8 chainlets (there are two residues per chain) on the convex surface of LRD structures, form two 3- Chain β-pleated sheet and 1 2- chain β-pleated sheet.There is no spiral in TSHR LRD structures, as shown in Figure 4.Use David Keith Smith (1989, undisclosed data) is based on DSSP algorithms (Kabsch W, Sander C 1983Dictionary of proteinsecondary structure：pattern recognition of hydrogen-bonded andgeometrical features.Biopolymers 22：2677-2637) the SSTRUC softwares identification two level knot of exploitation Structure.Upper all five (N77, N99, N113, the Nl77 and Nl98) glycosylation sites of TSHR are all located at convex surface.

TSHR-M22 Fab compounds

The structure of TSHR-M22 Fab compounds shows that M22 Fab are combined with TSHR 260 concave surface, and the TSRH Interface of the symmetry axis of " pipe " almost between autoantibody light and weight chain is parallel, as shown in Figure 5.Compared to be not bound with M22 that (rmsd of all atoms is 0.4 to a little residues), most of residue positioned at the combination interface of M22 antibody variable region exists Same position is nearly at when being attached to TSHR.For HC P97 C_αAtom, it was observed that the atom of M22 trunk residues is most Large deviation only has 1.1In addition, compared with uncombined M22, the side chain of only 6 M22 residues, which exists, is more than 2Deviation (table 5).

The difference of the overall positions of M22 Fab constant regions and the overall positions of uncombined M22 Fab structures is around perseverance Determining the axle between area and variable region has about 20 degree of rotation, and the rotation is due to caused by Molecule in Crystal is accumulated.There are enough electricity Carbon aquation of the sub- density to a glycosylation site (N26) on acceptor on all 5 possible glycosylation site and M22 Fab Compound residue is modeled.All glycosylation sites on TSHR260 all away from combination interface, do not influence M22 Fab knots Close.The comparative result of TSHR260-M22 Fab compounds and FSH-FSH LRD compounds is shown in Fig. 6.Fig. 6 shows two kinds of structures Animation figure, using only FSHR and TSHR residue carry out it is overlapping.

Some aspects that arrangement is combined in compound especially allow people surprised, and these aspects include：

(a) M22 clasps TSHR LRD concave surface in a manner of being very similar to FSH and clasp TSHR LRD.Moreover, M22 2 solid axles (2 fold axis) and FSH 2 solid axles are completely overlapped in their own compound.It is apparent that autoantibody with Hormone has almost identical binding characteristic.Prior art is not on this prompting.

(b) it is in addition, very big by (2500 with the TSHR LRD of M22 interactions recessed surface area), and extend from N-terminal To C-terminal.

This is surprising and unexpected, and prior art is not prompted this extensive interaction.

(c) in addition to the large area combined with LRD, in complex crystal structure observe different types of phase The intensity and quantity of interaction are surprising.For example, there is the net of 22 hydrogen bonds and salt bridge between the albumen of two interactions Network is most rare.

(d) comparison to the M22 in compound and free M22 structure shows, participates in the M22 residues that TSHR is combined Atom does not move substantially.This also allows people surprised, because some induced-fits anticipated that originally.When FSH and FSHR is tied Also there is obvious movement during conjunction.

On the TSHR LRD structures of itself, it is shockingly similar with FSHR LRD, but also has some significant differences, bag Include the quantity of β chains.

Autoantibody-acceptor interaction

Share 2500Solvent accessible surface is buried in the interface between autoantibody and acceptor.TSHR and Interaction between M22Fab represents an extensive hydrogen bond network, salt bridge (22 hydrogen bonds and salt bridge), non-hydrogen bond polarity phase Interaction and hydrophobic contact (table 2；Fig. 7 and Fig. 8) mixing.In fig. 8, the residue of interaction is represented and marked with rod Go out.Hydrogen bond is represented by dashed line.

Although two chains all form a large amount of hydrogen bonds and salt bridge (heavy chain 14, light chain 8), with TSHR interactions M22 Fab heavy chain residues are more than light chain.The residue of most of M22 Fab interactions is located at Kabat (Kabat E, Perry H, Wu T, Gottesman K, Foeller C1991 Sequences of proteins of immunological Interest, 5th ed.US PublicService Health Service.Bethesda, MD) define hypervariable region L2, H1, H2 and H3, as depicted in fig. 5e.In Fig. 5 e, participate in the residue that acceptor combines and (Otwinowski is all represented with runic and underscore The Processing of X-ray diffraction datacollected in oscillation of Z, Minor W 1997 mode.Methods in Enzymology：MacromolecularCrystallography, part A 276：307-326). Bury the surface at the interface between TSHR LRD and M22LC, TSHR LRD for 526.9And M22 LC for 526.5And for the interaction between TSHR LRD and M22HC, TSHR LRD area is 730.1And M22 HC's Area is 730.4

There are 7 hydrogen bonds between TSHR LRD and M22 LC, also there are 7 hydrogen bonds between TSHR LRD and M22 HC.It is specific and Speech, M22 HC Y99 and two TSHR residues El07 (including M22 Y99 trunk nitrogen and TSHR E107 side chain) and K58 (side chain for including the two residues) is with Hydrogenbond.M22LC Q53 also produce two hydrogen bonds (with TSHR N208 and Q235). 3 hydrogen bonds of the TSHR K129 generations including M22 HC T30 and T53, and TSHR Q235 and 2 M22 residues (LC D52 With LC Q53) with Hydrogenbond.

TSHR260-M22 Fab interactions also include the hydrogen bond (being shown in Table 2) of 14 water mediations.Participate in M22 with strong model (i.e. interactive surfaces product is more than 60 for De Huali interactions) TSHR residues be R255 (102.2)、R80(91.9)、R38(76.6 )、K129(75.5 )、K183(64.4 ) and R109 (60.6).Participate in TSHR260 with (i.e. interactive surfaces product is more than 70 for strong Van der Waals force interaction) M22 residues be HC R28 (115.8)、HC Y56(86.6 )、LC Y50(86.4 )、HC Y99(79.4 )、LC Q53(76.7 ) and HC P97 (70.8).Existing electrostatic interaction is related to following residue in TSHR260-M22 Fab compounds (using intensity decreases as sequence)： TSHR D1 51 and M22 HC R28 (minimum ranges 2.76), TSHR K58 and M22 LC D95A (minimum ranges 2.60 ), TSHR R80 and M22 HC D54 (minimum ranges 2.75), TSHR K183 and M22 HC E96 (minimum ranges 3.04 ), TSHR K209 and M22 LC D52 (minimum ranges 3.57), TSHR R80 and M22 HC D52 (minimum ranges 3.20 ), TSHR K129 and M22 HC R28 (minimum ranges 4.05), TSHR K209 and M22 HC D51 (minimum ranges 4.50 ), TSHR R255 and M22 LC D60 (minimum ranges 4.39), TSHRK129 and M22 LC D52 (minimum ranges 5.27 ) (table 3), these Henderson-Hasselbalch equatioies determinations for passing through and being used to calculate residue side chains electric charge that interact, and And performing in assessing the self-compiling program of the distance between atomic charge and charge atom, pH to be accounted for into it In.

In TSHR residues, TSHR R80 and M22 residues produce most strong accumulation electrostatic interaction, and M22 HC R28 with TSHR residues produce most strong accumulation electrostatic interaction.It is negative that hypervariable region H1, H2 and H3 of M22 Fab heavy chains residue form band The outer rim of electric cavity, the negatively charged cavity region phase highly positively charged with TSHR (R38, K58, R80, Hl05, K129) Interaction.

Mutation in TSHR LRD is to M22 to having with the interaction seen in TSHR260-M22 Fab crystal structures The influence for the stimulation that CAMP produces in the expression TSHR of pass Chinese hamster ovary celI

The action effect for the ring AMP activity that the amino acid mutation in TSHR LRD by testing measure is stimulated M22 is such as Described in WO2006/016121A.Then these effects are analyzed according to the interaction found in TSHR260-M22 crystal structures Effect.

For example, mutation R80A, E107A, R109A, K129A, K183A, Y185A, R255A have significantly to M22 stimulating activities Influence (60%) (WO2006/016121A) lower than wild type TSHR activity, and TSHR260 and M22 in complex crystal structure Transactional analysis between Fab shows that all these TSHR residues interact (table 2 and 3) with M22 Fab.Moreover, it Participate in the effects (table 2) of 9 in the structure in 22 hydrogen bonds and salt bridge, and TSHR R109 generate the hydrogen of two water mediations Key (table 2) and the interaction of strong Van der Waals force.R80, E107, R109, K183, Y185 and R255 also assist in two non-hydrogen bond polarity Interaction and the hydrophobic contact (table 2) with M22.

Another mutation being had a significant impact as described in WO2006/016121A in TSHR LRD to M22 excitant activity It is F130A, and crystal structure shows the F130 with M22 HC P97 and HCG98 (table 2) hydrophobic contact.

Several new mutation (not described in WO2006/016121A) have been carried out in TSHR LRD and it has been carried out Test.Mutation K209E has a significant impact (be less than wild type TSHR activity 20%) to M22 stimulating activities.TSHR K209 join With the non-hydrogen bond polar interaction with M22LC Y50, hydrophobic contact, and and M22LC are formed with M22 LC Q53 (table 2) For D51 and LC D52 (table 4) formed with electrostatic interaction is attracted, why this can cause M22 loss of activity to mutation T SHR K209E (be less than wild-type activity TSHR 20%) provides explanation.

Turn with the mutation couple TSHR relevant with the interaction seen in TSHR260-M22 Fab crystal structures in M 22 The action effect that transfected cho cell middle ring AMP M22 is stimulated

Several Single amino acid mutations are introduced into M22 Fab heavy chains and sequence of light chain, and have studied these mutation to expression The action effect (table 4) that the M22 that cyclic adenosine monophosphate generates in wild type TSHR Chinese hamster ovary celI is stimulated.

It is several in mutation when performance in the effective amino acid of M22 stimulating activities the 80% of wild type (be less than), HC R28 and TSHR D151 form 3 salt bridges, and polar interaction is formd with TSHR F153 and I152, and with TSHR I152 and F153 forms hydrophobic contact (table 2).Strong electrostatic interaction, and and TSHRH105 occur for M22HC D52 and TSHR R80 Generation polar interaction (table 2 and 3).Strong electrostatic interaction occurs for M22 HC D54 and TSHR R80, and passes through water With TSHR T104 with Hydrogenbond (table 2 and 3), polar interaction, and and TSHR occur for M22 HC Y56 and TSHR R38 Strong hydrophobic contact (table 2) occurs for R38, T56 and R80.Finally, M22 LC D52 and TSHR Q235 formation hydrogen bond, and with Strong electrostatic interaction (table 2 and 3) occurs for TSHR K209.

By in the analysis of influence result of the mutation in TSHR and M22 to M22 stimulating activities and its complex crystal structure The analysis of interaction between TSHR and M22 Fab combines, and is causing bioactivity in recognizable TSHR and M22 Fab Interaction on be critically important residue.Specifically, TSHR R38 and M22 HL T57 Hydrogenbonds, and TSHR R80 with M22 Fab (HC D52 and HC D54) form 3 salt bridges, and participate in the hydrophobic contact (table 2) with M22 HC Y56.In addition, TSHR K129 and M22 Fab (HC T30 and HC T53) form 3 hydrogen bonds.Therefore, the N- ends band of TSHR LRD concave surfaces is being just The residue cluster of electric charge is critically important (Fig. 5 d) in the M22 interactions with causing bioactivity.

However, our experiment also found bioactivity of the residue for M22 of the C-terminal part of concave surface in TSHR LRD Also it is critically important, and participate in the interaction in complex crystal structure between TSHR and M22.In these residues, TSHR R255 It is particularly subject to pay close attention to, because R255 mutation has a significant impact to M22 activity, but TSH activity is not influenceed (WO2006/016121A).Several interactions occur for TSHR R255 and M22 Fab in crystal structure (with M22 LC V58 shapes Into two hydrogen bonds, electrostatic interaction is formed with M22 LC D60, polar interaction is formed with LC D60, and with LC L54 shapes Into hydrophobic interaction) (see above and table 2 and 3).

It is involved in and the interaction of the TSHR LRD in compound (table 2) in many of M22 heavy chains and light chain residue. There is (M22 HC R28, HC D52, HC D54, HC Y56 and LC D52) to be particularly subject to pay close attention among these, because such as us Shown in experiment, the mutation of these residues has influence on M22 bioactivity (see upper and table 2 and 4).

Conclusion

In a word, analysis of the various mutation in TSHR or M22 to the effect of M22 stimulating activities and TSHR260-M22 Fab Crystal structure in observe correlation between there is good uniformity.This shows the TSHR260 compound with M22 Fab The method that crystal structure provides design molecular structure, point of the molecular structure by being interacted with TSHR or with such as M22 Son by disturb the interaction of acceptor-autoantibody it is this in a manner of interact.This interference effect provides a kind of pre- The method of the TSHR autoantibody stimulations of anti-GravesShi patients.

For example, design come fill the small molecule of the negatively charged cavity in M22 surfaces (being formed by M22 H1, H2 and H3) should M22 (and TSHR autoantibodies with similarity surface feature) is prevented to be attached on acceptor, wherein the negatively charged sky Chamber interacts with the highly positively charged ridge in the N- ends of TSHR LRD concave surfaces.On the contrary, design come with above-mentioned TSHR LRD The small molecule of positively charged ridge interaction is expected and can prevent M22 (and the TSHR itself with similarity surface feature is anti- Body) interacted with TSRH.In addition, small molecule can be designed to prevent important TSRH residues R255 and M22 and other TSRH itself Antibody interacts.

Specific amino acid mutation in TSHR LRD and M22 can be designed to study TSHR activation mechanisms, thus point out into The method that one step prevents TSHR autoantibodies activation TSHR.Moreover, the reason to TSHR activation mechanisms obtained from these researchs Solution can provide research and understand the method for gonadotropic hormone receptor activation, because gonadotropic hormone acceptor and close phase in TSHR structures Close.

Moreover, being understood in detail to the interaction between the M22 and TSHR that are provided by complex crystal structure, in conjunction with The further research of above mentioned receptor binding mechanisms, can design the recruit as TSHR activators.When containing TSHR Tissue when needing to be stimulated, this thyroid gland stimulation molecule can work in vivo.For example, it is used to stimulate as current Any residual thyroid cancer left after ablation¹³¹The people of I intakes recombinates TSH substitute.

The details that TSHR-M22 crystal structures provide also allow the part for designing new improvement to be used for measuring and assessing TSHR autoantibodies in patient serum sample.

Interaction between TSHR and M22 is complicated extensively.Moreover, with FSH (Fan QR, Hendrickson WA 2005 Structure of human follicle-stimulating hormone incomplex with its receptor.Nature 433：269-277) comparison of compound FSHR crystal structure and TSHR-M22 crystal structures shows, M22 relative TSHR in a manner of FSHR relative with FSH positioning is about the same are positioned itself (table 6).Specifically, M22 and FSH exist Respective acceptor is clasped relative to about 90 ° of directions of receptor tube major axis.TSH-TSHR interaction comparison model show, by The compound that FSH and its acceptor are formed has extremely similar structure (Nunez with the compound being compounded to form by FSH and FSHR Miguel R, Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Blundell TL, The Analysis of the thyrotropin receptor- of Rees Smith B, Furmaniak J 2004 thyrotropininteraction by comparative modeling.Thyroid 14：991-1011 and NunezMiguel R, Sanders J, Blundell TL, Rees Smith B3 Furmaniak J 2005Comparative Modeling of the Thyrotropin Receptor.Thyroid 15：746-747).

Immune system and TSHR evolution pressure are meaningful, and it causes the formation of autoantibody, the autoantibody By simulating TSH effect with acceptor interaction, wherein similar with hormone with the interaction mode of acceptor.Now, exist The details of M22-TSHR interactions is clearly determined on molecular level, why these evolution pressures and TSHR can be gone out Some understandings of existing autoimmunity may be apparent from.

Table 1.2.55Crystal data under resolution ratio is collected and statistics of refining

Space group I2₁2₁2₁

Structure cell：A, b, c () 43.89,175.78,205.81

Resolving range () 30.0-2.55(2.61-2.55)

Rsym¹%) 7.1 (36.1)

Integrality (%) 96.1 (99.2)

Special reflection quantity 25,731

Average redundancy 4.6

Mean intensity,<I/σ(I)> 10.5

During %I/ σ (I) ＞ 3 47.5 under highest resolution scope

Reflection

The Wilson B- factors () 47.7

Resolving range () 26.7-2.55

Reflect quantity：Work/test 23,125/1301

Rcryst²(%) 18.1

R_free ³(%) 24.5

Non-hydrogen atom quantity：

Albumen 5,039

2-Acetamido-2-deoxy-D-glucose 84

Zn²⁺ 5

Water 289

Estimate location data error⁴() 0.30

Deviation key root-mean-square-deviation () 0.009

Deviation angle root-mean-square-deviation () 1.236

Ramachandran plot is analyzed⁵ 36.0

Residue quantity：

Allow position 561

It is unrestricted to allow position 2

Position 2 is not allowed

3.1 Crystal data under resolution ratio is collected and statistics of refining

Space group I2₁2₁2₁

Structure cell：A, b, c () 43.73,175.16,204.66

Resolving range () 30.0-3.10(3.17-3.10)

Rsym¹(%) 8.0 (40.4)

Integrality (%) 99.4 (99.8)

Special reflection quantity 15,037

Average redundancy 4.2

Mean intensity,<I/σ(I)> 8.7

During %I/ σ (I) ＞ 3 42.9 under highest resolution scope

Reflection

The Wilson B- factors () 66.2

Resolving range () 28.3-3.10

Reflect quantity：Work/test 13,267/734

Rcryst²(%) 20.7

Rfree³(%) 28.3

Non-hydrogen atom quantity：

Albumen 5,027

2-Acetamido-2-deoxy-D-glucose 84

Zn²⁺ 9

Water 38

Estimate location data error⁴() 0.54

Deviation key root mean square () 0.006

Deviation angle root mean square () 1.019

Ramachandran plot is analyzed⁵ 46.0

Residue quantity：

Allow position 558

It is unrestricted to allow position 4

Position 1 is not allowed

Value represents the corresponding statistics under highest resolution scope in round parentheses.

¹R_sym=∑ | I_h-<I>|/∑_hI_h, I here_hIt is the intensity for reflecting h,<I>It is being averaged for all relative symmetry reflections Intensity.

²R_cryst=∑ ‖ F_obs|-|F_calc|/∑|F_obs|, F_obsAnd F_calcActual measurement and the structure factor amplitude calculated.

³R_freeFor R_crystUse the random subset of data (the about 5%) (BrungerAT 1992 excluded outside refining Free R value：a novel statistical quantity for assessing theaccuracy of crystal structures.Nature 355：472-475).

⁴Estimate location data error, the R calculated based on REFMAC_freeIt is worth (Murshudov GN, VaginAA, Dodson EJ 1997 Refinement of macromolecular structures by themaximum-likelihood method.Acta Crystallograpy SelectionD53：240-255).

⁵(Laskowski RA, MacArthur MW, Moss DS, Thornton JM 1993 is calculated with PROCHECK PROCHECK：a program to check the stereochemicalquality of protein structures.J Appl Crystallogr 26：283-281).

Interaction between TSHR260 the and M22 Fab that table 2. is observed in the crystal structure of compound

* salt bridge is represented.

The hydrogen bond of water mediation

¹Hydrogen bond distance range is in 2.3-3.4Between.

²Letter in bracket represents that M22 Fab chain residues belong to：A- light chains, B- heavy chains.

³The distance of polarity contact is between 3.4 to 4.0Between.

⁴Carbon carbon is contacted 4.0It is interior.

Table 3.

Ion Thermodynamic parameters in TSHR260-M22 Fab compounds

The shown interaction strength for comparing, unit is newton, using self-compiling program (ELECINT, R.Nunez Miguel, unpublished) calculate, wherein ε=1 is taken to Electrostatic field calculation, to using Henderson- The pendant atom electric charge that Hasselbalch formula calculate charged residues takes pH=7.4.

The influence that the M22 that cyclic adenosine monophosphate generates in Chinese hamster ovary celI of the mutation to expressing wild type TSHR in table 4.M22 is stimulated

The M22 Fab preparations of mutation	M22 Fab are to stimulation caused by cAMP
		Wild type	+++++
HC R28D	+++
		HC T30A	+++++
HC D52A	++
		HC D52K	-
HC D54R	+
		HC Y56A	++
HC K64E	+++++
		HC K73D	+++++
HC R94E	+++
		HC E96A	++++
HC E96R	Expression is not detected
		LC D51K	+++++
LC D52A	+++
		LC D52R	Expression is not detected
LC D93R	+++++

+++ ++=wild-type activity (100%),

The 100-80% of +++ +=＜ wild-type activities,

The 80-60% of +++=＜ wild-type activities,

The 60-40% of ++=＜ wild-type activities,

The 40-20% of +=＜ wild-type activities,

The 20% of -=＜ wild-type activities.

The effect of ability that M22 and mark that every kind of mutation suppresses mark to M22 Fab TSH are combined with TSHR and to ring The stimulation of adenylate is similar.

Table 5.

The deviation of atom site in uncombined M22 and the M22 structures of combination

Trunk

The change that can be uniquely considered on trunk is：

Pro97HC, its C alpha atom occur 1.1Skew

Side chain

Skew (is more than 2)

Arg28HC, its NH2 atom occur 4.8Skew.

Trp33HC, its NE1 atom occur 4.0Skew.

Arg66LC, its NH1 atom occur 3.2Skew.

Lys64HC, its NZ atom occur 3.0Skew.

Asp95LC, its OD1 atom occur 2.2Skew.

Asp 52HC, its OD1 atom occur 2.1Skew.

Skew (is more than 1.3And less than 2)

Ser56LC, its OG atom occur 1.8Skew.

Tyr99HC, its OH atom occur 1.6Skew.

Asp ó O LC, its OD2 atom occur 1.4Skew.

Pro97HC, its CB atom occur 1.3Skew.

Table 6.

The comparison of FSHR-FSH and TSHR-M22 compounds

A. respectively with FSH or M22 Fab (HC=heavy chains；LC=light chains) with the FSHR or TSHR of Van der Waals force interaction Residue.

B. respectively with FSH or M22 Fab (HC=heavy chains；LC=light chains) it is residual with the FSHR of interaction of hydrogen bond or TSHR Base.

C. respectively with FSH or M22 Fab (HC=heavy chains；LC=light chains) with the FSHR of strong ion Thermodynamic parameters or TSHR residues.

Residue numbering in Table A-C corresponds to the residue of FSHR and TSHR sequence same positions.

A. Van der Waals force interacts

^*Produce strong interaction residue ()。

B. hydrogen bond

X³The residue produces three hydrogen bonds with corresponding chain

C. ion Thermodynamic parameters (interaction strength is more than 4.0e-10N)

X²Represent two ion Thermodynamic parameters, X³Represent three interactions.

<110>R S R Co., Ltds

<120>The crystal structure of THSR acceptors

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Arg Ile Pro Ser Leu Pro Pro Ser Thr Gln Thr Leu Lys Leu Ile Glu

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Thr His Leu Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn

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Ile Ser Arg Ile Tyr Val Ser Ile Asp Val Thr Leu Gln Gln Leu Glu

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Ser His Ser Phe Tyr Asn Leu Ser Lys Val Thr His Ile Glu Ile Arg

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Asn Thr Arg Asn Leu Thr Tyr Ile Asp Pro Asp Ala Leu Lys Glu Leu

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Pro Leu Leu Lys Phe Leu Gly Ile Phe Asn Thr Gly Leu Lys Met Phe

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Pro Asp Leu Thr Lys Val Tyr Ser Thr Asp Ile Phe Phe Ile Leu Glu

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Ile Thr Asp Asn Pro Tyr Met Thr Ser Ile Pro Val Asn Ala Phe Gln

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Gly Leu Cys Asn Glu Thr Leu Thr Leu Lys Leu Tyr Asn Asn Gly Phe

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Thr Ser Val Gln Gly Tyr Ala Phe Asn Gly Thr Lys Leu Asp Ala Val

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Tyr Leu Asn Lys Asn Lys Tyr Leu Thr Val Ile Asp Lys Asp Ala Phe

180 185 190

Gly Gly Val Tyr Ser Gly Pro Ser Leu Leu Asp Val Ser Gln Thr Ser

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Val Thr Ala Leu Pro Ser Lys Gly Leu Glu His Leu Lys Glu Leu Ile

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Ala Arg Asn Thr

225

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Phe Ser Gly Phe Gly Asp Leu Glu Lys Ile Glu Ile Ser Gln Asn Asp

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Val Leu Glu Val Ile Glu Ala Asp Val Phe Ser Asn Leu Pro Lys Leu

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His Glu Ile Arg Ile Glu Lys Ala Asn Asn Leu Leu Tyr Ile Asn Pro

85 90 95

Glu Ala Phe Gln Asn Leu Pro Asn Leu Gln Tyr Leu Leu Ile Ser Asn

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Thr Gly Ile Lys His Leu Pro Asp Val His Lys Ile His Ser Leu Gln

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Lys Val Leu Leu Asp Ile Gln Asp Asn Ile Asn Ile His Thr Ile Glu

130 135 140

Arg Asn Ser Phe Val Gly Leu Ser Phe Glu Ser Val Ile Leu Trp Leu

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Asn Lys Asn Gly Ile Gln Glu Ile His Asn Cys Ala Phe Asn Gly Thr

165 170 175

Gln Leu Asp Glu Leu Asn Leu Ser Asp Asn Asn Asn Leu Glu Glu Leu

180 185 190

Pro Asn Asp Val Phe His Gly Ala Ser Gly Pro Val Ile Leu Asp Ile

195 200 205

Ser Arg Thr Arg Ile His Ser Leu Pro Ser Tyr Gly Leu Glu Asn Leu

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Lys Lys Leu Arg Ala Arg Ser Thr Tyr Asn Leu Lys Lys Leu Pro Thr

225 230 235 240

Leu Glu

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Met Arg Pro Ala Asp Leu Leu Gln Leu Val Leu Leu Leu Asp Leu Pro

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Arg Asp Leu Gly Gly Met Gly Cys Ser Ser Pro Pro Cys Glu Cys His

20 25 30

Gln Glu Glu Asp Phe Arg Val Thr Cys Lys Asp Ile Gln Arg Ile Pro

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Ser Leu Pro Pro Ser Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu

50 55 60

Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg

65 70 75 80

Ile Tyr Val Ser Ile Asp Val Thr Leu Gln Gln Leu Glu Ser His Ser

85 90 95

Phe Tyr Asn Leu Ser Lys Val Thr His Ile Glu Ile Arg Asn Thr Arg

100 105 110

Asn Leu Thr Tyr Ile Asp Pro Asp Ala Leu Lys Glu Leu Pro Leu Leu

115 120 125

Lys Phe Leu Gly Ile Phe Asn Thr Gly Leu Lys Met Phe Pro Asp Leu

130 135 140

Thr Lys Val Tyr Ser Thr Asp Ile Phe Phe Ile Leu Glu Ile Thr Asp

145 150 155 160

Asn Pro Tyr Met Thr Ser Ile Pro Val Asn Ala Phe Gln Gly Leu Cys

165 170 175

Asn Glu Thr Leu Thr Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val

180 185 190

Gln Gly Tyr Ala Phe Asn Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn

195 200 205

Lys Asn Lys Tyr Leu Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val

210 215 220

Tyr Ser Gly Pro Ser Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala

225 230 235 240

Leu Pro Ser Lys Gly Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn

245 250 255

Thr Trp Thr Leu Lys Lys Leu Pro Leu Ser Leu Ser Phe Leu His Leu

260 265 270

Thr Arg Ala Asp Leu Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn

275 280 285

Gln Lys Lys Ile Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser

290 295 300

Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser

305 310 315 320

Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly

325 330 335

Tyr Lys Glu Lys Ser Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr

340 345 350

Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln

355 360 365

Glu Leu Lys Asn Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His

370 375 380

Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro

385 390 395 400

Lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr Lys Phe

405 410 415

Leu Arg Ile Val Val Trp Phe Val Ser Leu Leu Ala Leu Leu Gly Asn

420 425 430

Val Phe Val Leu Leu Ile Leu Leu Thr Ser His Tyr Lys Leu Asn Val

435 440 445

Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Ala Asp Phe Cys Met Gly

450 455 460

Met Tyr Leu Leu Leu Ile Ala Ser Val Asp Leu Tyr Thr His Ser Glu

465 470 475 480

Tyr Tyr Asn His Ala Ile Asp Trp Gln Thr Gly Pro Gly Cys Asn Thr

485 490 495

Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu

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Thr Val Ile Thr Leu Glu Arg Trp Tyr Ala Ile Thr Phe Ala Met Arg

515 520 525

Leu Asp Arg Lys Ile Arg Leu Arg His Ala Cys Ala Ile Met Val Gly

530 535 540

Gly Trp Val Cys Cys Phe Leu Leu Ala Leu Leu Pro Leu Val Gly Ile

545 550 555 560

Ser Ser Tyr Ala Lys Val Ser Ile Cys Leu Pro Met Asp Thr Glu Thr

565 570 575

Pro Leu Ala Leu Ala Tyr Ile Val Phe Val Leu Thr Leu Asn Ile Val

580 585 590

Ala Phe Val Ile Val Cys Cys Cys Tyr Val Lys Ile Tyr Ile Thr Val

595 600 605

Arg Asn Pro Gln Tyr Asn Pro Gly Asp Lys Asp Thr Lys Ile Ala Lys

610 615 620

Arg Met Ala Val Leu Ile Phe Thr Asp Phe Ile Cys Met Ala Pro Ile

625 630 635 640

Ser Phe Tyr Ala Leu Ser Ala Ile Leu Asn Lys Pro Leu Ile Thr Val

645 650 655

Ser Asn Ser Lys Ile Leu Leu Val Leu Phe Tyr Pro Leu Asn Ser Cys

660 665 670

Ala Asn Pro Phe Leu Tyr Ala Ile Phe Thr Lys Ala Phe Gln Arg Asp

675 680 685

Val Phe Ile Leu Leu Ser Lys Phe Gly Ile Cys Lys Arg Gln Ala Gln

690 695 700

Ala Tyr Arg Gly Gln Arg Val Pro Pro Lys Asn Ser Thr Asp Ile Gln

705 710 715 720

Val Gln Lys Val Thr His Asp Met Arg Gln Gly Leu His Asn Met Glu

725 730 735

Asp Val Tyr Glu Leu Ile Glu Asn Ser His Leu Thr Pro Lys Lys Gln

740 745 750

Gly Gln Ile Ser Glu Glu Tyr Met Gln Thr Val Leu

755 760

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Ser Leu Pro Pro Ser Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu

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Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg

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Asn Glu Thr Leu Thr Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val

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Gln Gly Tyr Ala Phe Asn Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn

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Lys Asn Lys Tyr Leu Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val

210 215 220

Tyr Ser Gly Pro Ser Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala

225 230 235 240

Leu Pro Ser Lys Gly Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn

245 250 255

Thr Trp Thr Leu Lys Lys Leu Pro Leu Ser Leu Ser Phe Leu His Leu

260 265 270

Thr Arg Ala Asp Leu Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn

275 280 285

Gln Lys Lys Ile Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser

290 295 300

Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser

305 310 315 320

Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly

325 330 335

Tyr Lys Glu Lys Ser Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr

340 345 350

Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln

355 360 365

Glu Leu Lys Asn Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His

370 375 380

Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro

385 390 395 400

Lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr Lys Phe

405 410 415

Leu Arg Ile Val Val Trp Phe Val Ser Leu Leu Ala Leu Leu Gly Asn

420 425 430

Val Phe Val Leu Leu Ile Leu Leu Thr Ser His Tyr Lys Leu Asn Val

435 440 445

Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Ala Asp Phe Cys Met Gly

450 455 460

Met Tyr Leu Leu Leu Ile Ala Ser Val Asp Leu Tyr Thr His Ser Glu

465 470 475 480

Tyr Tyr Asn His Ala Ile Asp Trp Gln Thr Gly Pro Gly Cys Asn Thr

485 490 495

Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu

500 505 510

Thr Val Ile Thr Leu Glu Arg Trp Tyr Ala Ile Thr Phe Ala Met Arg

515 520 525

Leu Asp Arg Lys Ile Arg Leu Arg His Ala Cys Ala Ile Met Val Gly

530 535 540

Gly Trp Val Cys Cys Phe Leu Leu Ala Leu Leu Pro Leu Val Gly Ile

545 550 555 560

Ser Ser Tyr Ala Lys Val Ser Ile Cys Leu Pro Met Asp Thr Glu Thr

565 570 575

Pro Leu Ala Leu Ala Tyr Ile Val Phe Val Leu Thr Leu Asn Ile Val

580 585 590

Ala Phe Val Ile Val Cys Cys Cys Tyr Val Lys Ile Tyr Ile Thr Val

595 600 605

Arg Asn Pro Gln Tyr Asn Pro Gly Asp Lys Asp Thr Lys Ile Ala Lys

610 615 620

Arg Met Ala Val Leu Ile Phe Thr Asp Phe Ile Cys Met Ala Pro Ile

625 630 635 640

Ser Phe Tyr Ala Leu Ser Ala Ile Leu Asn Lys Pro Leu Ile Thr Val

645 650 655

Ser Asn Ser Lys Ile Leu Leu Val Leu Phe Tyr Pro Leu Asn Ser Cys

660 665 670

Ala Asn Pro Phe Leu Tyr Ala Ile Phe Thr Lys Ala Phe Gln Arg Asp

675 680 685

Val Phe Ile Leu Leu Ser Lys Phe Gly Ile Cys Lys Arg Gln Ala Gln

690 695 700

Ala Tyr Arg Gly Gln Arg Val Pro Pro Lys Asn Ser Thr Asp Ile Gln

705 710 715 720

Val Gln Lys Val Thr His Glu Met Arg Gln Gly Leu His Asn Met Glu

725 730 735

Asp Val Tyr Glu Leu Ile Glu Lys Ser His Leu Thr Pro Lys Lys Gln

740 745 750

Gly Gln Ile Ser Glu Glu Tyr Met Gln Thr Val Leu

755 760

<210>18

<211>763

<212>PRT

<213>People (Homo sapiens)

<400>18

Met Arg Pro Ala Asp Leu Leu Gln Leu Val Leu Leu Leu Asp Leu Pro

1 5 10 15

Arg Asp Leu Gly Gly Met Gly Cys Ser Ser Pro Pro Cys Glu Cys His

20 25 30

Gln Glu Glu Asp Phe Arg Val Thr Cys Lys Asp Ile Gln Arg Ile Pro

35 40 45

Ser Leu Pro Pro Ser Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu

50 55 60

Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg

65 70 75 80

Ile Tyr Val Ser Ile Asp Val Thr Leu Gln Gln Leu Glu Ser His Ser

85 90 95

Phe Tyr Asn Leu Ser Lys Val Thr His Ile Glu Ile Arg Asn Thr Arg

100 105 110

Asn Leu Thr Tyr Ile Asp Pro Asp Ala Leu Lys Glu Leu Pro Leu Leu

115 120 125

Lys Ser Leu Ala Phe Ser Asn Thr Gly Leu Lys Met Phe Pro Asp Leu

130 135 140

Thr Lys Val Tyr Ser Thr Asp Ile Phe Phe Ile Leu Glu Ile Thr Asp

145 150 155 160

Asn Pro Tyr Met Thr Ser Ile Pro Val Asn Ala Phe Gln Gly Leu Cys

165 170 175

Asn Glu Thr Leu Thr Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val

180 185 190

Gln Gly Tyr Asp Phe Phe Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn

195 200 205

Lys Asn Lys Tyr Leu Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val

210 215 220

Tyr Ser Gly Pro Ser Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala

225 230 235 240

Leu Pro Ser Lys Gly Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn

245 250 255

Ser Trp Thr Leu Lys Lys Leu Ala Leu Ser Leu Ser Phe Leu His Leu

260 265 270

Thr Arg Ala Asp Leu Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn

275 280 285

Gln Lys Lys Ile Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser

290 295 300

Ser Ile Glu Thr Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser

305 310 315 320

Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly

325 330 335

Tyr Lys Glu Lys Ser Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr

340 345 350

Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln

355 360 365

Glu Leu Lys Asn Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His

370 375 380

Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro

385 390 395 400

Lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr Lys Phe

405 410 415

Leu Arg Ile Val Val Trp Phe Val Ser Leu Leu Ala Leu Leu Gly Asn

420 425 430

Val Phe Val Leu Leu Ile Leu Leu Thr Ser His Tyr Lys Leu Asn Val

435 440 445

Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Ala Asp Phe Cys Met Gly

450 455 460

Met Tyr Leu Leu Leu Ile Ala Ser Val Asp Leu Tyr Thr His Ser Glu

465 470 475 480

Tyr Tyr Asn His Ala Ile Asp Trp Gln Thr Gly Pro Gly Cys Asn Thr

485 490 495

Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu

500 505 510

Thr Val Ile Thr Leu Glu Arg Trp Tyr Ala Ile Thr Phe Ala Met Ala

515 520 525

Leu Asp Arg Lys Ile Arg Leu Arg His Ala Cys Ala Ile Met Val Gly

530 535 540

Gly Trp Val Cys Cys Phe Leu Leu Ala Leu Leu Pro Leu Val Gly Ile

545 550 555 560

Ser Ser Tyr Ala Lys Val Ser Ile Cys Leu Pro Met Asp Thr Glu Thr

565 570 575

Pro Leu Ala Leu Ala Tyr Ile Val Phe Val Leu Thr Leu Asn Ile Val

580 585 590

Ala Phe Val Ile Val Cys Cys Cys Tyr Val Lys Ile Tyr Ile Thr Val

595 600 605

Arg Asn Pro His Asn Pro Gly Asp Lys Asp Thr Lys Ile Ala Lys Arg

610 615 620

Met Ala Val Leu Ile Phe Thr Asp Phe Thr Cys Met Ala Pro Ile Ser

625 630 635 640

Phe Tyr Ala Val Ser Ala Ile Leu Asn Lys Pro Leu Ile Thr Val Ser

645 650 655

Asn Ser Lys Ile Leu Leu Val Leu Phe Tyr Pro Ile Asn Ser Cys Ala

660 665 670

Asn Pro Phe Leu Tyr Ala Ile Phe Thr Lys Ala Phe Gln Arg Asp Val

675 680 685

Phe Ile Leu Leu Ser Lys Phe Gly Ile Cys Lys Arg Gln Ala Gln Ala

690 695 700

Tyr Arg Gly Gln Arg Val Pro Pro Lys Asn Ser Thr Asp Ile Gln Val

705 710 715 720

Gln Lys Val Thr His Asp Met Arg Gln Gly Leu His Asn Met Glu Asp

725 730 735

Val Tyr Glu Leu Ile Glu Asn Ser His Leu Thr Pro Lys Lys Gln Gly

740 745 750

Gln Ile Ser Glu Glu Tyr Met Gln Thr Val Leu

755 760

<210>19

<211>764

<212>PRT

<213>People (Homo sapiens)

<400>19

Met Arg Pro Ala Asp Leu Leu Gln Leu Val Leu Leu Leu Asp Leu Pro

1 5 10 15

Arg Asp Leu Gly Gly Met Gly Cys Ser Ser Pro Pro Cys Glu Cys His

20 25 30

Gln Glu Glu Asp Phe Arg Val Thr Cys Lys Asp Ile Gln Arg Ile Pro

35 40 45

Ser Leu Pro Pro Ser Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu

50 55 60

Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg

65 70 75 80

Ile Tyr Val Ser Ile Asp Val Thr Leu Gln Gln Leu Glu Ser His Ser

85 90 95

Phe Tyr Asn Leu Ser Lys Val Thr His Ile Glu Ile Arg Asn Thr Arg

100 105 110

Asn Leu Thr Tyr Ile Asp Pro Asp Ala Leu Lys Glu Leu Pro Leu Leu

115 120 125

Lys Phe Leu Gly Ile Phe Asn Thr Gly Leu Lys Met Phe Pro Asp Leu

130 135 140

Thr Lys Val Tyr Ser Thr Asp Ile Phe Phe Ile Leu Glu Ile Thr Asp

145 150 155 160

Asn Pro Tyr Met Thr Ser Ile Pro Val Asn Ala Phe Gln Gly Leu Cys

165 170 175

Asn Glu Thr Leu Thr Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val

180 185 190

Gln Gly Tyr Ala Phe Asn Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn

195 200 205

Lys Asn Lys Tyr Leu Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val

210 215 220

Tyr Ser Gly Pro Ser Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala

225 230 235 240

Leu Pro Ser Lys Gly Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn

245 250 255

Thr Trp Thr Leu Lys Lys Leu Pro Leu Ser Leu Ser Phe Leu His Leu

260 265 270

Thr Arg Ala Asp Leu Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn

275 280 285

Gln Lys Lys Ile Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser

290 295 300

Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser

305 310 315 320

Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly

325 330 335

Tyr Lys Glu Lys Ser Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr

340 345 350

Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln

355 360 365

Glu Leu Lys Asn Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His

370 375 380

Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro

385 390 395 400

Lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr Lys Phe

405 410 415

Leu Arg Ile Val Val Trp Phe Val Ser Leu Leu Ala Leu Leu Gly Asn

420 425 430

Val Phe Val Leu Leu Ile Leu Leu Thr Ser His Tyr Lys Leu Asn Val

435 440 445

Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Ala Asp Phe Cys Met Gly

450 455 460

Met Tyr Leu Leu Leu Ile Ala Ser Val Asp Leu Tyr Thr His Ser Glu

465 470 475 480

Tyr Tyr Asn His Ala Ile Asp Trp Gln Thr Gly Pro Gly Cys Asn Thr

485 490 495

Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu

500 505 510

Thr Val Ile Thr Leu Glu Arg Trp Tyr Ala Ile Thr Phe Ala Met Arg

515 520 525

Leu Asp Arg Lys Ile Arg Leu Arg His Ala Cys Ala Ile Met Val Gly

530 535 540

Gly Trp Val Cys Cys Phe Leu Leu Ala Leu Leu Pro Leu Val Gly Ile

545 550 555 560

Ser Ser Tyr Ala Lys Val Ser Ile Cys Leu Pro Met Asp Thr Glu Thr

565 570 575

Pro Leu Ala Leu Ala Tyr Ile Val Phe Val Leu Thr Leu Asn Ile Val

580 585 590

Ala Phe Val Ile Val Cys Cys Cys His Val Lys Ile Tyr Ile Thr Val

595 600 605

Arg Asn Pro Gln Tyr Asn Pro Gly Asp Lys Asp Thr Lys Ile Ala Lys

610 615 620

Arg Met Ala Val Leu Ile Phe Thr Asp Phe Ile Cys Met Ala Pro Ile

625 630 635 640

Ser Phe Tyr Ala Leu Ser Ala Ile Leu Asn Lys Pro Leu Ile Thr Val

645 650 655

Ser Asn Ser Lys Ile Leu Leu Val Leu Phe Tyr Pro Leu Asn Ser Cys

660 665 670

Ala Asn Pro Phe Leu Tyr Ala Ile Phe Thr Lys Ala Phe Gln Arg Asp

675 680 685

Val Phe Ile Leu Leu Ser Lys Phe Gly Ile Cys Lys Arg Gln Ala Gln

690 695 700

Ala Tyr Arg Gly Gln Arg Val Pro Pro Lys Asn Ser Thr Asp Ile Gln

705 710 715 720

Val Gln Lys Val Thr His Asp Met Arg Gln Gly Leu His Asn Met Glu

725 730 735

Asp Val Tyr Glu Leu Ile Glu Asn Ser His Leu Thr Pro Lys Lys Gln

740 745 750

Gly Gln Ile Ser Glu Glu Tyr Met Gln Thr Val Leu

755 760

<210>20

<211>764

<212>PRT

<213>People (Homo sapiens)

<400>20

Met Arg Pro Ala Asp Leu Leu Gln Leu Val Leu Leu Leu Asp Leu Pro

1 5 10 15

Arg Asp Leu Gly Gly Met Gly Cys Ser Ser Pro Pro Cys Glu Cys His

20 25 30

Gln Glu Glu Asp Phe Arg Val Thr Cys Lys Asp Ile Gln Arg Ile Pro

35 40 45

Ser Leu Pro Pro Ser Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu

50 55 60

Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg

65 70 75 80

Ile Tyr Val Ser Ile Asp Val Thr Leu Gln Gln Leu Glu Ser His Ser

85 90 95

Phe Tyr Asn Leu Ser Lys Val Thr His Ile Glu Ile Arg Asn Thr Arg

100 105 110

Asn Leu Thr Tyr Ile Asp Pro Asp Ala Leu Lys Glu Leu Pro Leu Leu

115 120 125

Lys Phe Leu Gly Ile Phe Asn Thr Gly Leu Lys Met Phe Pro Asp Leu

130 135 140

Thr Lys Val Tyr Ser Thr Asp Ile Phe Phe Ile Leu Glu Ile Thr Asp

145 150 155 160

Asn Pro Tyr Met Thr Ser Ile Pro Val Asn Ala Phe Gln Gly Leu Cys

165 170 175

Asn Glu Thr Leu Thr Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val

180 185 190

Gln Gly Tyr Ala Phe Asn Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn

195 200 205

Lys Asn Lys Tyr Leu Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val

210 215 220

Tyr Ser Gly Pro Ser Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala

225 230 235 240

Leu Pro Ser Lys Gly Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn

245 250 255

Thr Trp Thr Leu Lys Lys Leu Pro Leu Ser Leu Ser Phe Leu His Leu

260 265 270

Thr Arg Ala Asp Leu Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn

275 280 285

Gln Lys Lys Ile Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser

290 295 300

Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser

305 310 315 320

Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly

325 330 335

Tyr Lys Glu Lys Ser Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr

340 345 350

Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln

355 360 365

Glu Leu Lys Asn Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His

370 375 380

Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro

385 390 395 400

Lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr Lys Phe

405 410 415

Leu Arg Ile Val Val Trp Phe Val Ser Leu Leu Ala Leu Leu Gly Asn

420 425 430

Val Phe Val Leu Leu Ile Leu Leu Thr Ser His Tyr Lys Leu Asn Val

435 440 445

Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Ala Asp Phe Cys Met Gly

450 455 460

Met Tyr Leu Leu Leu Ile Ala Ser Val Asp Leu Tyr Thr His Ser Glu

465 470 475 480

Tyr Tyr Asn His Ala Ile Asp Trp Gln Thr Gly Pro Gly Cys Asn Thr

485 490 495

Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu

500 505 510

Thr Val Ile Thr Leu Glu Arg Trp Tyr Ala Ile Thr Phe Ala Met Arg

515 520 525

Leu Asp Arg Lys Ile Arg Leu Arg His Ala Cys Ala Ile Met Val Gly

530 535 540

Gly Trp Val Cys Cys Phe Leu Leu Ala Leu Leu Pro Leu Val Gly Ile

545 550 555 560

Ser Ser Tyr Ala Lys Val Ser Ile Cys Leu Pro Met Asp Thr Glu Thr

565 570 575

Pro Leu Ala Leu Ala Tyr Ile Val Phe Val Leu Thr Leu Asn Ile Val

580 585 590

Ala Phe Val Ile Val Cys Cys Cys Tyr Val Lys Ile Tyr Ile Thr Val

595 600 605

Arg Asn Pro Gln Tyr Asn Pro Gly Asp Lys Asp Thr Lys Ile Ala Lys

610 615 620

Arg Met Ala Val Leu Ile Phe Thr Asp Phe Ile Cys Met Ala Pro Ile

625 630 635 640

Ser Phe Tyr Ala Leu Ser Ala Ile Leu Asn Lys Pro Leu Ile Thr Val

645 650 655

Ser Asn Ser Lys Ile Leu Leu Val Leu Phe Tyr Pro Leu Asn Ser Cys

660 665 670

Ala Asn Pro Phe Leu Tyr Ala Ile Phe Thr Lys Ala Phe Gln Arg Asp

675 680 685

Val Phe Ile Leu Leu Ser Lys Phe Gly Ile Cys Lys Arg Gln Ala Gln

690 695 700

Ala Tyr Arg Gly Gln Arg Val Pro Pro Lys Asn Ser Thr Asp Ile Gln

705 710 715 720

Val Gln Lys Val Thr His Glu Met Arg Gln Gly Leu His Asn Met Glu

725 730 735

Asp Val Tyr Glu Leu Ile Glu Asn Ser His Leu Thr Pro Lys Lys Gln

740 745 750

Gly Gln Ile Ser Glu Glu Tyr Met Gln Thr Val Leu

755 760

<210>21

<211>764

<212>PRT

<213>People (Homo sapiens)

<400>21

Met Arg Pro Ala Asp Leu Leu Gln Leu Val Leu Leu Leu Asp Leu Pro

1 5 10 15

Arg Asp Leu Gly Gly Met Gly Cys Ser Ser Pro Pro Cys Glu Cys His

20 25 30

Gln Glu Glu Asp Phe Arg Val Thr Cys Lys Asp Ile Gln Arg Ile Pro

35 40 45

Ser Leu Pro Pro Ser Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu

50 55 60

Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg

65 70 75 80

Ile Tyr Val Ser Ile Asp Leu Thr Leu Gln Gln Leu Glu Ser His Ser

85 90 95

Phe Tyr Asn Leu Ser Lys Val Thr His Ile Glu Ile Arg Asn Thr Arg

100 105 110

Asn Leu Thr Tyr Ile Asp Pro Asp Ala Leu Lys Glu Leu Pro Leu Leu

115 120 125

Lys Phe Leu Gly Ile Phe Asn Thr Gly Leu Lys Met Phe Pro Asp Leu

130 135 140

Thr Lys Val Tyr Ser Thr Asp Ile Phe Phe Ile Leu Glu Ile Thr Asp

145 150 155 160

Asn Pro Tyr Met Thr Ser Ile Pro Val Asn Ala Phe Gln Gly Leu Cys

165 170 175

Asn Glu Thr Leu Thr Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val

180 185 190

Gln Gly Tyr Ala Phe Asn Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn

195 200 205

Lys Asn Lys Tyr Leu Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val

210 215 220

Tyr Ser Gly Pro Ser Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala

225 230 235 240

Leu Pro Ser Lys Gly Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn

245 250 255

Thr Trp Thr Leu Lys Lys Leu Pro Leu Ser Leu Ser Phe Leu His Leu

260 265 270

Thr Arg Ala Asp Leu Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn

275 280 285

Gln Lys Lys Ile Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser

290 295 300

Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser

305 310 315 320

Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly

325 330 335

Tyr Lys Glu Lys Ser Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr

340 345 350

Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln

355 360 365

Glu Leu Lys Asn Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His

370 375 380

Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro

385 390 395 400

Lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr Lys Phe

405 410 415

Leu Arg Ile Val Val Trp Phe Val Ser Leu Leu Ala Leu Leu Gly Asn

420 425 430

Val Phe Val Leu Leu Ile Leu Leu Thr Ser His Tyr Lys Leu Asn Val

435 440 445

Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Ala Asp Phe Cys Met Gly

450 455 460

Met Tyr Leu Leu Leu Ile Ala Ser Val Asp Leu Tyr Thr His Ser Glu

465 470 475 480

Tyr Tyr Asn His Ala Ile Asp Trp Gln Thr Gly Pro Gly Cys Asn Thr

485 490 495

Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu

500 505 510

Thr Val Ile Thr Leu Glu Arg Trp Tyr Ala Ile Thr Phe Ala Met Arg

515 520 525

Leu Asp Arg Lys Ile Arg Leu Arg His Ala Cys Ala Ile Met Val Gly

530 535 540

Gly Trp Val Cys Cys Phe Leu Leu Ala Leu Leu Pro Leu Val Gly Ile

545 550 555 560

Ser Ser Tyr Ala Lys Val Ser Ile Cys Leu Pro Met Asp Thr Glu Thr

565 570 575

Pro Leu Ala Leu Ala Tyr Ile Val Phe Val Leu Thr Leu Asn Ile Val

580 585 590

Ala Phe Val Ile Val Cys Cys Cys Tyr Val Lys Ile Tyr Ile Thr Val

595 600 605

Arg Asn Pro Gln Tyr Asn Pro Gly Asp Lys Asp Thr Lys Ile Ala Lys

610 615 620

Arg Met Ala Val Leu Ile Phe Thr Asp Phe Ile Cys Met Ala Pro Ile

625 630 635 640

Ser Phe Tyr Ala Leu Ser Ala Ile Leu Asn Lys Pro Leu Ile Thr Val

645 650 655

Ser Asn Ser Lys Ile Leu Leu Val Leu Phe Tyr Pro Leu Asn Ser Cys

660 665 670

Ala Asn Pro Phe Leu Tyr Ala Ile Phe Thr Lys Ala Phe Gln Arg Asp

675 680 685

Val Phe Ile Leu Leu Ser Lys Phe Gly Ile Cys Lys Arg Gln Ala Gln

690 695 700

Ala Tyr Arg Gly Gln Arg Val Pro Pro Lys Asn Ser Thr Asp Ile Gln

705 710 715 720

Val Gln Lys Val Thr His Asp Met Arg Gln Gly Leu His Asn Met Glu

725 730 735

Asp Val Tyr Glu Leu Ile Glu Asn Ser His Leu Thr Pro Lys Lys Gln

740 745 750

Gly Gln Ile Ser Glu Glu Tyr Met Gln Thr Val Leu

755 760

Claims (24)

1. one kind is by SEQ ID NO:The common knot that TSHR polypeptides and TSHR binding entities shown in 16 amino acid/11-260 are formed Crystalline substance, wherein the TSHR binding entities are M22, the cocrystallization belongs to orthogonal I2₁2₁2₁Space group, there is cell parameter Fig. 9 b of location data or cell parameter 43.73,175.16 and 204.66 shown in 43.89,175.78 and 205.81 Fig. 9 a Shown location data.

2. it is a kind of identify with TSHR interaction part method, methods described include identification with TSHR polypeptides three-dimensional structure or The part of at least one amino acid interaction of its homologue, the TSHR polypeptides three-dimensional structure or its homologue are computer The TSHR polypeptides three-dimensional structure or its homologue for the image that system is provided, wherein the computer system is for showing basis The computer system of the tomograph of the TSHR polypeptides of claim 1, the computer system include with corresponding diagram 9a or The data storage facility of the data of 9b TSHR polypeptide amino acid location datas.

3. method according to claim 2, wherein the electronic image of the TSHR polypeptides three-dimensional structure shows human TSHR polypeptide Rich leucine domain.

4. method according to claim 3, wherein the electronic image of the TSHR polypeptides three-dimensional structure at least shows mankind TSHR Amino acid 30-257.

5. method according to claim 2, wherein the part is TSHR activators.

6. method according to claim 2, wherein the part is TSHR antagonists.

7. method according to claim 2, wherein identify with described TSHR amino acid K129, E107, K58 and Y185 extremely Few one by forming hydrogen bond the part that interacts.

8. method according to claim 2, wherein identify with described TSHR amino acid residues R255, R80, K129, R38 and It is at least one by forming Van der Waals force interaction and the part of interaction in Kl83.

9. method according to claim 2, wherein identify with the TSHR amino acid residues D151, K58, K129, R80, It is at least one by forming electrostatic interaction the part that interacts in K209 and K183.

10. method according to claim 2, wherein identifying with the TSHR amino acid residues K209 by forming ion pair phase Interaction and the part to interact.

11. a kind of identify the method that may interfere with the part that autoantibody is combined with TSHR, methods described includes using computer The tomograph for the TSHR polypeptides that system is provided is identified on the M22 surfaces for being at least filled substantially with being made up of M22H1, H2 and H3 The part of negatively charged cavity, wherein the computer system is three for showing TSHR polypeptides according to claim 1 The computer system of structure chart is tieed up, the computer system includes the TSHR polypeptide amino acids positioning number with corresponding diagram 9a or 9b According to data data storage facility.

12. method according to claim 11, wherein the electronic image of the TSHR polypeptides three-dimensional structure shows that mankind TSHR is more The rich leucine domain of peptide.

13. method according to claim 12, wherein the electronic image of the TSHR polypeptides three-dimensional structure at least shows the mankind TSHR amino acid 30-257.

14. method according to claim 11, wherein the autoantibody is thyroid autoantibody.

15. method according to claim 11, wherein the autoantibody is TSH antagonists.

16. method according to claim 11, methods described includes identification and at least destroys TSHR residues R255 substantially with promoting first shape The part that gland autoantibody combines.

17. method according to claim 14, methods described includes identification and destroys mankind TSHR amino acid K209 and thyroid The part that autoantibody combines.

18. method according to claim 16, methods described includes identification and destroys mankind TSHR amino acid K209 and thyroid The part that autoantibody combines.

19. according to the method for claim 2 or 11, wherein measure part and the possible interaction of other acceptors.

20. method according to claim 19, wherein the possible interaction is to combine.

21. method according to claim 19, wherein other described acceptors include follicle-stimulating hormone acceptor and/or lutropin by Body.

22. according to the method for claim 2 or 11, wherein the computer system is used to provide and the portion of antibody or antibody thereon The TSHR polypeptides of split-phase interaction or the tomograph of its homologue.

23. according to the method for claim 2 or 11, wherein the computer system includes being used to show TSHR polypeptide three-dimensional structures The display of figure.

24. method according to claim 23, wherein the computer system is used to show and TSHR polypeptides or its homologue phase Antibody of interaction or part thereof.