Actin-induced dimerization of palladin
promotes actin-bundling
Ravi Vattepu, Rahul Yadav, and Moriah R. Beck*
Chemistry Department, Wichita State University, Wichita, Kansas 67260
Received 6 August 2014; Accepted 9 October 2014
DOI: 10.1002/pro.2588
Published online 11 October 2014 proteinscience.org
Abstract: A subset of actin binding proteins is able to form crosslinks between two or more actin filaments, thus producing structures of parallel or networked bundles. These actin crosslinking proteins
interact with actin through either bivalent binding or dimerization. We recently identified two binding
sites within the actin binding domain of palladin, an actin crosslinking protein that plays an important
role in normal cell adhesion and motility during wound healing and embryonic development. In this
study, we show that actin induces dimerization of palladin. Furthermore, the extent of dimerization
reflects earlier comparisons of actin binding and bundling between different domains of palladin. On
the basis of these results we hypothesized that actin binding may promote a conformational change
that results in dimerization of palladin, which in turn may drive the crosslinking of actin filaments. The
proximal distance between two actin binding sites on crosslinking proteins determines the ultrastructural properties of the filament network, therefore we also explored interdomain interactions
using a combination of chemical crosslinking experiments and actin cosedimentation assays. Limited
proteolysis data reveals that palladin is less susceptible to enzyme digestion after actin binding. Our
results suggest that domain movements in palladin are necessary for interactions with actin and are
induced by interactions with actin filaments. Accordingly, we put forth a model linking the structural
changes to functional dynamics.
Keywords: actin; crosslinking; dimerization; palladin; bundling
Introduction
Abbreviations: ABP, actin binding protein; BS3, bis(sulfosuccinimidyl)suberate; CH, calponin homology; DFDNB, 1,5-difluoro2,4-dinitrobenzene; DSP, dithiobis(succinimidylpropionate); DTT,
dithiothreitol; ECM, extracellular matrix; F-actin, filamentous
actin; FH2, formin homology domain; ICL, intramolecular
crosslinked; Ig, immunoglobulin; IPTG, isopropyl b-D-1-thiogalactopyranoside; Kd, dissociation constant; OD, optical density; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis; TEV, tobacco etch virus; Vt, vinculin tail
domain.
Additional Supporting Information may be found in the online
version of this article.
Grant sponsor: Start-up funds from Wichita State University and
a Center of Biomedical Research Excellence (COBRE) grant
from the National Center for Research Resources; Grant number: 5P20RR017708; Grant sponsor: The National Institute of
General Medical Sciences; Grant number: 8P20GM103420;
Grant sponsor: Institutional Development Award (IDeA) from the
National Institute of General Medical Sciences from the National
Institutes of Health; Grant number: P20GM103418.
*Correspondence to: Moriah R. Beck, 1845 Fairmount St., Wichita
State University, Wichita, KS. E-mail: moriah.beck@wichita.edu
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Actin regulates diverse cellular processes including
cell division, adhesion, motility, and vesicle trafficking through dynamic turnover between monomeric
(G-actin) and filamentous (F-actin) forms. Actin
binding proteins (ABPs) are responsible for constructing a wide array of filament assemblies that
range from meshworks of individual crosslinked
actin filaments to closely spaced bundles of F-actin.1
The architecture of these assemblies is largely dictated by the type of actin crosslinking protein and
its spatial and temporal regulation.2 Assemblies of
loosely spaced and orthogonally connected threedimensional crosslinked actin filaments are present
in the cellular cortex to support the cell membrane,
lamellipodium, and membrane ruffles. These structures provide mechanical strength and shape as well
as fluidity to irreversible deformation of the cell.3,4
Likewise actin bundles can be tightly or loosely
packed into parallel actin filaments that provide
mechanical support to microvilli, stereocilia, and
C 2014 The Protein Society
Published by Wiley-Blackwell. V
filopodia for their specialized functions.5,6 The architecture of filament meshworks is also determined by
the binding affinity and stoichiometry of the crosslinking protein involved.7 The distinction between
actin crosslinking and bundling activities of ABPs is
complicated in some cases because the ability to
transform actin filaments from a weakly crosslinked
complex into a tightly bundled network is partially
determined by the their local concentration, which is
only evident in in vitro assays. Espin, fascin, and aactinin, for example, all form crisscrossed arrays of
actin filaments at low crosslinker concentrations
that evolve into tightly bundled filaments at higher
crosslinker concentrations.8–10 Nonetheless, other
classes of crosslinking proteins only produce isotropically crosslinked networks at any crosslinker
ratio.
The actin binding protein palladin is ubiquitous
in developing and adult vertebrate tissues and localizes in actin rich structures such as stress fibers,
focal adhesions, podosomes, and Z-discs.11 Many
recent studies suggest that deregulation of palladin
expression may play a key role in the invasive cell
motility that characterizes metastatic cancer cells as
well as in the development of cardiovascular diseases.12–15 Localization of palladin in invadopodia
and filopodia alongside other actin organizing proteins in these structures suggests that palladin is an
important player in both normal and invasive motility. Despite the growing correlative evidence linking
palladin to increased invasiveness, anti-migratory
functions for actin bundling by palladin have also
been reported.16 Because of this lack of clarity
regarding the precise role of palladin in cancer progression, our goal was to determine the specific actin
crosslinking mechanism that palladin employs to
enable a better understanding of the dynamic coordination between the cytoskeleton and tumor cell
invasion.
Palladin belongs to a novel subfamily that
includes two structurally related proteins, myotilin
and myopalladin, that are present in skeletal and
heart muscle, respectively, and all three are characterized by multiple immunoglobulin (Ig) domains
[Fig. 1(A)]. Similar Ig domains are also found in
other cytoskeletal organizing proteins including
myomesin, titin, myBP-C, and myBP-H, where they
maintain the structural integrity of the sarcomere.17,18 Crosslinking of F-actin was initially only
associated with the tandem Ig3–4 domain of palladin. However, while characterizing the actin-binding
functions of the Ig3 domain, Beck et al. observed
that the Ig3 domain could also crosslink F-actin at
higher concentrations than employed in initial
experiments.19 Beck et al. also identified mutations
to two basic patches on the surface of the Ig3
domain that are involved in binding actin. It was
inferred that crosslinking of actin filaments could
Vattepu et al.
Figure 1. No evidence of homodimerization from chemical
crosslinking of palladin domains. A: Schematic representation
of palladin’s largest (200 kDa) isoform, which includes immunoglobulin (Ig) domains 1 through 5 and two polyproline
regions represented by triangles. The brackets demarcate the
most abundant isoform (90 kDa) of palladin that incudes only
the second polyproline region to the Cterminus. The Ig3–4
domains of palladin are highlighted with the dashed square. B–
E: Various palladin domains (7.5 mM) were incubated with varying concentrations of the chemical crosslinker (15–30 mM) for
the indicated times, ranging from 10 to 40 min. Reaction products were subsequently analyzed for crosslinking on SDSPAGE. Crosslinking of Ig3 (B), Ig4 (C), Ig3–4 (D) with DFDNB
does not show any dimer or other oligomeric species. E: Ig3–4
chemical crosslinking with BS3 reveals the formation of an
intramolecular crosslinked monomer (denoted by star) that
migrates faster than the monomeric species in SDS gels.
occur at these two sites, however this does not rule
out the possibility that palladin self-associates to
promote actin bundling.
Homodimerization is a common mechanism for
proteins with small actin-binding domains to bind
two filaments simultaneously. In fact, the palladinrelated protein myotilin forms dimers via two Ig
domains that are homologous to palladin’s Ig
PROTEIN SCIENCE VOL 24:70—80
71
domains 4 and 5.20 Involvement of regions outside
the actin-binding domain cannot be ruled out, as the
tandem Ig3–4 domain of palladin shows significantly
higher binding and bundling activity than the isolated Ig3 domain.19 Moreover, Chin and Toker have
identified an Akt1-mediated phosphorylation of a
specific serine residue (S507) in the linker region
between the Ig 3 and 4 domains of palladin that
seems to affect not only migration of breast cancer
cells but also the in vivo bundling activity of
palladin.16
This highlights the importance for determining
the mechanism by which the actin-binding domain
of palladin is able to modulate actin filament architecture. In contrast to villin and a-actinin that can
both self-associate even in the absence of actin, our
group has shown that Ig3 domain remains monomeric in the absence of actin.19 Therefore we wanted
to determine whether palladin uses a crosslinking
mechanism similar to vinculin, VASP, and scruin
whereby actin binding results in homodimerization.21 We have employed chemical crosslinking
techniques to determine the role of the different palladin Ig domains in actin crosslinking as well as the
role actin itself plays in controlling the activity of
palladin. These results shed light on the mechanism
whereby actin-binding induces dimerization of palladin, which in turn affects the phase transition of
actin from single filaments to bundles.
Results
Palladin immunoglobulin domains exist as
monomers in the absence of actin
Our previous study revealed that the Ig3 domain of
palladin has two F-actin binding sites, suggesting that
bundling of F-actin occurs through binding at these
two sites.19 However, actin bundling by the tandem
Ig3–4 domain is much more robust than Ig3 alone,
resulting in more bundles at lower concentrations of
palladin.19 One explanation is that the Ig4 domain of
palladin may be involved in dimerization to enhance
actin bundling. We hypothesized that the F-actin bundling activity of Ig3, as well as the enhanced bundling
by Ig3–4 domains of palladin, may be due to the selfassociation of palladin. Therefore, we wanted to determine whether the palladin domains (Ig3, Ig4, and/or
Ig3–4) have any propensity to form homodimers in
solution in the absence of actin. Previous sedimentation equilibrium measurements indicated that these
domains have similar apparent and predicted molecular weights, which suggests that these domains exist
as monomers in solution;19 however, these experiments
were carried out at micromolar concentrations of protein and therefore may not be able to detect weak oligomeric species.
We have used the chemical crosslinkers DFDNB
and BS3 to detect oligomerization of the Ig3, Ig4,
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and Ig3–4 domains in the absence of actin. First, we
used DFDNB with reactive fluorine groups that
form stable aryl-amine bonds with nearby amine
groups. Our results in Figure 1(B–D) show that neither of the C-terminal Ig domains of palladin form
any high molecular weight oligomeric species in the
absence of actin under moderately stringent crosslinking conditions (7.5 mM protein, 30 mM crosslinker, and 40 min reaction time). Increased
crosslinker concentrations and reaction times can
induce minor dimerization in the absence of actin,
which likely do not represent a biologically relevant
dimeric state. Interestingly, when Ig3–4 was allowed
to react with the BS3 crosslinker in the absence of
actin, a significant fraction of protein was found in
an intramolecular crosslinked monomer [Fig. 1(E)],
which migrates faster than native Ig3–4 in a SDSPAGE gel. We do not see this intramolecular crosslinked species of Ig3–4 in the presence of DFDNB
[Fig. 1(D)], which is likely due to the shorter spacer
arm length of DFDNB (3.0 Å) as compared with BS3
(11.4 Å). This suggests that both of the Ig domains
in Ig3–4 maintain a close distance in the absence of
actin.
Actin induces dimerization in the actin-binding
domain of palladin
Previous studies with the vinculin tail (Vt) domain
indicate that actin can play an active role in modulating its own filament organization by inducing oligomerization.22,23 Therefore, we next employed
chemical crosslinking studies to ascertain whether
dimerization of the Ig3, Ig4, and Ig3–4 domains of
palladin occurs in the presence of actin. DFDNB
crosslinking of the Ig3 domain in presence of actin
reveals two different oligomeric crosslinked species,
corresponding to a 24 kDa Ig3 homodimer and a 54
kDa Ig3:actin heterodimer [Fig. 2(A)]. The composition of these homo- and hetero-dimeric species, as
well as other high molecular weight oligomers of Ig3
and actin, were confirmed by probing the Western
blots with both palladin and actin antibodies [Fig.
3(A)].
Chemical crosslinking of Ig3–4 with DFDNB in
the presence of actin likewise reveals Ig3–4 homodimers of 53 kDa as well as 69 kDa heterodimers
[Fig. 2(D)], also confirmed by immunoblotting [Fig.
3(B)]. Homodimeriztion of Ig3 requires greater concentrations of protein as compared to the Ig3–4 actin
induced dimerization. These results indicate that
the Ig3 domain is sufficient for actin-induced dimerization, just as it is sufficient for both binding and
bundling. However, the tandem Ig3–4 domain has a
lower threshold for dimerization, which recapitulates the increased bundling activity of Ig3–4 that
was previously observed.24
Similar crosslinking experiments with the isolated Ig4 domain do not show any dimer in the
Actin-Induced Dimerization of Paladin
used the DFDNB crosslinker in a mixture of equal
concentrations of Ig3 and Ig4 domains in the presence of F-actin, with reaction conditions identical to
the tandem Ig3–4 domains. Our results for the mixture of Ig3 and Ig4 reveal actin-induced dimers of
Ig3 at 24 kDa and 54 kDa [Fig. 2(C)] that are similar in quantity to previous actin-induced Ig3 oligomerization in absence of Ig4 [Fig. 2(A)]. Moreover
actin cosedimentation assays with a mixture of Ig3
and Ig4 revealed that actin binding is not higher
than Ig3 alone (data not shown); suggesting that the
free Ig4 domain in solution does not affect Ig3 binding to F-actin. Another possible explanation is that
the linker region between Ig3 and Ig4 orients the
Ig4 domain favorably during actin bundling and
thereby exposes a second dimerization site on Ig4.
This conformation could manifest itself only in the
presence of the tandem Ig3–4 domains.
Chemically crosslinked products were also sedimented at high speed to separate the actin bound
and free fractions of palladin. Palladin Ig3–4 homodimers cosediment exclusively with the actin filaments [Fig. 4(A), pellet lanes]. This finding is
consistent with the idea that actin binding induces
palladin dimerization and suggests that the homodimeric form of palladin is functionally active [Fig.
4(A)]. To determine how the concentration of actin
effects palladin dimerization, we performed chemical
crosslinking reactions where the concentration of
Ig3–4 was held constant while varying the actin concentration (1.25–20 lm) in the presence of a chemical crosslinker. Our results show that even very low
concentrations of F-actin can induce palladin dimerization [Fig. 4(B)]. This substantiates the proposition
that actin has a critical role in dimerization of palladin’s actin binding domain.
Interdomain flexibility of Ig3–4 is important for
F-actin binding and bundling
Figure 2. Palladin homodimerizes in presence of actin. Palladin domains (5–10 mM) were incubated with F-actin (10 mM)
and varying concentrations of the chemical crosslinker
DFDNB (30–100 mM) for 20/40 min. Reaction products were
analyzed for crosslinking by SDS-PAGE. A: Ig3, B: Ig4, C: Ig3
1 Ig4, D: Ig3–4 chemical crosslinking with DFDNB. Palladin
homodimers (arrowhead) and palladin:actin heterodimers
(star) are observed in Ig3, Ig3 1 Ig4, and Ig3–4 as indicated.
The size of various dimers correspond to: Ig3 homodimer, 24
kDa; Ig3:actin, 54 kDa; Ig3–4 homodimer, 53 kDa; and Ig3–
4:actin heterodimer, 69 kDa.
presence of actin, which is most likely due to inability of Ig4 to interact directly with actin [Fig. 2(B)].
However, the presence of the Ig4 domain in tandem
with the Ig3 domain has shown much greater bundling of F-actin compared to Ig3 alone, suggesting a
yet unknown role of Ig4 in enhancing actin crosslinking activity. To examine the role of Ig4, we have
Vattepu et al.
We further explored the possibility of changes in the
interdomain interactions and the importance of the
flexible linker region between the Ig3 and Ig4
domains using a series of chemical crosslinking
experiments. As we have shown in Figure 1(E),
crosslinking of the tandem Ig3–4 domain by BS3
induces a significant fraction of Ig3–4 to form an
intramolecular crosslinked species in the absence of
actin. To determine the conformational flexibility
and/or role of the tandem domains in the presence of
actin, we have used BS3 crosslinking of Ig3–4 in
presence of actin under three different conditions.
First, Ig3–4 and BS3 crosslinker were both added to
polymerized actin at the same time to establish
whether intramolecular crosslinking of monomeric
Ig3–4 is favorable in the presence of F-actin [Fig.
5(A), Lane 1]. In the second condition, Ig3–4 was
first crosslinked with BS3 in the absence of F-actin.
This crosslinking reaction was quenched to prevent
PROTEIN SCIENCE VOL 24:70—80
73
Figure 3. Western blots confirm the presence of both homo- and heterodimeric species upon chemical crosslinking of palladin in
the presence of F-actin. Samples from the previous experiment [Fig. 2(A,D)] were probed against palladin and actin antibodies to
identity the species formed during chemical crosslinking reactions. DFDNB crosslinking reactions containing (A) 10 mM Ig3 1 10
mM actin and (B) 5 mM Ig3–4 1 10 mM actin with either the palladin or actin antibody alongside the molecular weight ladder.
any further crosslinking and then followed by addition of F-actin, thereby giving ample time for Ig3–4
to form intramolecular crosslinked species before
actin binding [Fig. 5(A), Lane 2]. In the final condition, Ig3–4 was incubated first with F-actin for 60
min and then BS3 crosslinker was added, giving the
least opportunity for Ig3–4 to form intramolecular
crosslinked species [Fig. 5(A), Lane 3]. Our results
Figure 4. Homodimers of palladin present in actin bound fraction and at very low concentrations of actin. A: Purified palladin
domains (10 mM) were incubated with F-actin (10 mM) and
DFDNB before being subjected to a high-speed centrifugation
(100,000g). Pellets (P) and supernatants (S) and were analyzed
on SDS-PAGE. The Ig3–4 dimer (star) and actin:Ig3–4 heterodimer (arrowhead) were observed in the pellet fraction only. B:
The Ig3–4 domain of palladin was incubated with DFDNB and
varying actin concentrations (1–20 mM) for 1 h. Reaction products were then analyzed on SDS-PAGE. Both the Ig3–4 homodimer (star) and actin:Ig34 heterodimer (arrowhead) were
observed at all concentrations of F-actin.
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show that Ig3–4 homodimers and Ig3–4:actin heterodimers are still present when F-actin is available for
interaction before crosslinker was added [Fig. 5(A)].
Moreover, the quantity of intramolecular crosslinked
Ig3–4 species varied significantly under different
conditions, suggesting different positions of the Ig4
domain with respect to Ig3 in the presence versus
absence of actin [Fig. 5(B)]. In conditions where Ig3–
4 was crosslinked before actin binding, the absence
of any Ig3–4:actin heterodimer combined with the
increased amount of intramolecular crosslinked species also suggest that domain movement within Ig3–
4 is necessary or is induced when palladin interacts
with F-actin.
To further explore the importance of flexibility
between the Ig3–4 domains we have used the crosslinker DSP, which is similar to BS3 in its spacer
arm length but has the advantage of a disulfide
bond in the spacer arm that can be cleaved upon
reduction. We first carried out an actin cosedimentation assay with Ig3–4 in the presence of
DSP without any reducing agent, which resulted in
formation of the intramolecular crosslinked species.
Furthermore this intramolecular crosslinked species
of Ig3–4 caused a reduction in the amount of actin
binding and bundling [Fig. 5(C,D)]. This reduction
could be due to the intramolecular crosslinked structural confinement of Ig3–4 or may result from the
chemical modification of lysine residues, for lysine
residues in palladin have been shown to be involved
in actin binding and bundling.19 Actin binding and
bundling activity were both partially restored by
cleaving the spacer arm of DSP, suggesting that the
loss of actin bundling and binding is the result of
intramolecular crosslinking and does not result from
chemical modifications of lysine residues. Our
results also indicate that actin bundling is abolished
completely in the absence of reducing agent,
Actin-Induced Dimerization of Paladin
Figure 5. Intramolecular crosslinking of palladin Ig3–4
domains displays cause and effect relationship with actin
binding and bundling. A: Lane 1 contains Ig3–4 (10 mM), BS3
(30 mM), and F-actin (10 mM) that were incubated together for
1 h. Lane 2 is Ig3–4 treated with BS3 for 1 h at RT in the
absence of F-actin to allow the formation of intramolecular
crosslinked monomers (star), followed by a 1 h incubation
with F-actin. Lane 3 is Ig3–4 incubated with F-actin for 1 h,
before addition of BS3. B: Quantification of the intramolecular
crosslinked (ICL) Ig3–4 species from lanes in A. C and D:
Ig3–4 was treated with the thiol-cleavable BS3 analog DSP
for 1 h in the absence of F-actin to yield intramolecularly
crosslinked monomers (ICL). Following a 30 min incubation in
either the absence or the presence of 50 mM DTT (C), the
binding of DSP treated Ig3–4 to Factin was assessed by a
co-sedimentation assay followed by SDS-PAGE and quantification of Ig3–4 band by densitometry. D: Bundling of F-actin
by DSP treated Ig3–4 was also assessed by a differential
sedimentation assay and the actin bands were quantified in
similar manner. The percentage of actin present in each fraction is represented as supernatant (light gray), pellet (dark
gray), and bundle (black).
whereas actin binding is only diminished [Fig.
5(C,D)]. This dichotomy suggests that interdomain
flexibility is more important for bundling and suggests that actin-binding induces a conformational
change in Ig3–4 that leads to the exposure of an
additional dimerization site on Ig3 or Ig4. This additional binding site could then enhance homodimeriztion of Ig3–4 to a greater degree than for Ig3 and
provides an explanation for the previous results that
revealed that Ig3–4 is a better actin bundling protein than Ig3.
Actin induced structural changes in Ig3–4 are
revealed by limited proteolysis
We carried out limited proteolysis experiments with
Ig3–4 in the presence and absence of F-actin to
Vattepu et al.
determine whether Ig3–4 undergoes a conformational change upon binding F-actin. First, we performed proteolysis of Ig3–4 with trypsin and
chymotrypsin at varying incubation times and protease concentrations, and our results show that the
rate of degradation is greater for trypsin as compared to chymotrypsin (Supporting Information, Fig.
S1). Furthermore we found that in the absence of Factin, Ig3–4 is more susceptible to enzyme digestion
than in the actin bound form (Fig. 6). Trypsin completely digested Ig3–4 in the supernatant within 20
min, whereas actin-bound Ig3–4 was not completely
digested until 60 min. The amount of intact Ig3–4
that remained after proteolysis was also quantified
and reveals that 60% of actin-bound Ig3–4 remains
after 20 min [Fig. 6(C,D)]. On the other hand, Ig3–4
that is not bound to actin was nearly completely
digested after only 10 min [Fig. 6(A,B)]. These
results suggest that actin binding by Ig3–4 causes a
change in conformation or orientation of the two
domains that causes protection from proteolysis. We
also performed proteolysis of Ig3 under similar conditions, however, we did not observe any change in
extent of proteolysis between free Ig3 and actinbound Ig3 (Supporting Information Fig. S2). Ig3
remained quite stable even when we employed
higher concentrations of both the proteases, and furthermore binding of Ig3 to F-actin does not appear
to involve in any detectable change in conformation.
We therefore suggest that this actin-induced conformational change most likely occurs in the tandem
Ig3–4 domain and results in a new orientation of the
two domains relative to each other.
Discussion
The general understanding from previous study
with a variety of crosslinking proteins is that the
crosslinking activity results from more than one
actin binding site. This can be achieved either
through two structurally related but different binding sites on the same polypeptide or one binding site
on each polypeptide that are then brought together
through oligomerization. The proximity of these
binding sites appears to correlate with the architecture of the resulting actin filament network. Two
proximal actin-binding sites, in fimbrin and in the
tandem calponin homology (CH) domains of fascin,
both result in tightly packed parallel bundles of
actin filaments.25,26
Non-covalent dimerization is another a common
actin crosslinking mechanism. For example, villin
has an N-terminal dimerization site that regulates
self-association, with or without actin, but is also
critical for bundling actin filaments.27,28 In a similar
fashion, the formin family of proteins homodimerize
and bundle actin filaments via their FH2 domain.29
Homodimers of filamin, where C-terminal self-association and N-terminal actin binding sites are
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Figure 6. Actin binding protects palladin from proteolysis. The Ig3–4 domain of palladin (50 mg) was incubated with F-actin (50
mg) and then centrifuged at 100,000g to separate the actin bound (pellet) and free (supernatant) fractions of Ig3–4. Both fractions were then subjected to limited proteolysis by using trypsin (1:1000) before separation and detection of fragments by SDSPAGE (A and B). The percentage of intact Ig3–4 (arrowhead) found in the supernatant (A and C) or actin bound pellet (B and D)
was quantitated by densitometry measurements of corresponding gels.
distantly located, provide flexibility to crosslink distant actin filaments and assemble into a loose and
orthogonal network.30,31 Similarly, crosslinking of
actin filaments by a-actinin is facilitated by the central rod domain of spectrin repeats that form an
antiparallel dimer, while N-terminal CH domains
provide a pair of actin binding sites.32 To understand
the network mechanics and organization of the actin
cytoskeleton, it is imperative to determine the binding interactions and structural role of the growing
list of ABPs using well-defined in vitro biochemical
techniques.
In this study, we have demonstrated the dimerization of palladin’s actin binding domain can be
induced upon binding to actin, which translates into
bundling of actin filaments. Palladin domains (Ig3,
Ig4, and Ig3–4) do not self-associate in the absence
of actin, as revealed by our chemical crosslinking
experiments with DFDNB crosslinker and also confirmed in previous studies of palladin oligomerization.19,22,27 Intramolecular crosslinked species were
observed with Ig3–4 in the presence of crosslinker
(BS3, 11.4 Å) with a longer spacer arm length than
DFDNB (3.0 Å); suggesting that domains in Ig3–4
may exist in an anti-parallel, “U” shaped orientation
connected through the 41 residue linker (discussed
more thoroughly in later section).
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The homodimers of Ig3 and Ig3–4 that we
observed only in the presence of actin (Fig. 2) suggest that palladin utilizes a mechanism whereby two
Ig3 or tandem Ig3–4 domains self-associate only
upon binding to actin in order to crosslink two actin
filaments. In contrast to a-actinin, utropin, and filamin, where actin binding and dimerization activities
reside in two separate domains;31–33 the Ig3 domain
of palladin seems to possess both an actin binding
site and at least one dimerization site that represents the minimum requisite for actin bundling
activity. Having different actin binding and dimerization domains separated by a flexible linker, as
observed in various other ABPs, alters the structural and viscoelastic properties of the crosslinked
actin filaments. Filamin, for example, contains more
linker domains than a-actinin and this results in
actin filament structures that are characterized by
actin bundles that have more viscoelasticity than
those formed by a-actinin.34 The viscoelastic properties of actin filament structures formed by the isolated Ig3 and Ig3–4 domains have not been studied
yet, however, the rheological properties of full length
palladin-actin networks have been found to be similar to those formed by a-actinin.35 Conventional wisdom would suggest that since actin binding and
dimerization sites reside in the same domain (Ig3),
Actin-Induced Dimerization of Paladin
Figure 7. Model for actin bundling and palladin Ig3–4 self-association. 1: Dimerization of palladin requires a conformational
change to take place, shown here as a loop displacement. Dimers of palladin may sporadically form but they are unstable and
the equilibrium is shifted toward the monomeric form. 2: Palladin binds to F-actin through the Ig3 domain, which results in conformational change that favors dimerization. 2 and 3: Upon dimerization, palladin promotes interaction between actin filaments
and their subsequent zippering into tight bundles. 4: Palladin-induced bundles could then elongate by polymerization of crosslinked actin filaments (4) and, although not determined yet, it is possible that palladin assembles both unipolar and mixed polarity bundles.
then palladin should form a stiffer actin network
than those formed by a-actinin. On the contrary, our
results suggest that actin bundling by palladin is
not a straightforward single domain mechanism as
the Ig3–4 domain displays far greater bundling and
actin-induced dimerization capabilities.
Although this study has focused on these two
domains, it has implications for full-length palladin
as all but one of the seven isoforms contain the Ig3
and Ig4 domains. It is important to note that other
regions of palladin are involved either directly or
indirectly in actin binding because the binding affinity of full-length palladin is significantly greater (Kd
2 mM) than the Ig3 domain (Kd 60 mM).24 This
difference in actin binding affinity is also evident
from the difference in intensity of homodimer protein bands present in the case of Ig3 versus the
more prevalent Ig3–4, when similar protein and
actin concentrations are used (Fig. 2). This reduction
in homodimer formation for the single domain could
likewise result from the lower actin binding affinity
of Ig3 (Kd 60 mM) as compared to Ig3–4 (Kd 9
mM), which would require an increase in the Ig3:actin molar ratio to saturate binding and cause more
substantial actin bundling. Distinct homodimers and
higher actin binding affinity were observed with
Ig3–4 and this can also be correlated with significantly higher bundling of actin filaments, as shown
even at lower Ig3–4/actin molar ratios (Fig. 5).
Despite the fact that the isolated Ig4 domain of
palladin has no direct interaction with actin, this
domain enhances the binding affinity of tandem
Ig3–4 by an unknown mechanism. On the basis of
our results, we put forth a mechanism to explain
this increased actin binding and crosslinking
Vattepu et al.
whereby a conformational change occurs in Ig3–4
that is induced by actin binding. In this model, the
conformational change leads to (1) the exposure of
either second binding or dimerization site on the Ig4
domain or (2) exposure of an otherwise cryptic actin
binding site on the Ig3 domain or linker between Ig3
and Ig4 domains that enhances the affinity of Ig3
for actin (Fig. 7).
Intramolecular crosslinked species that migrate
faster in SDS-PAGE were only observed in samples
of Ig3–4 containing the crosslinkers with a longer
spacer-arm length (BS3 and DSP), which suggests
that the orientation of the actin-bound form of Ig3–4
domain assumes a more extended form than the free
form. We found that the presence of intramolecular
crosslinked species inversely correlates with Ig3–4
bundling activity. This indicates that interactions
between actin and Ig3–4 inhibit intramolecular
crosslinking, which results in a more open conformation of the Ig3–4 domain [Fig. 5(A,B)]. Probing this
phenomenon further with the reversible DSP crosslinker revealed that the decreased actin binding and
bundling caused by intramolecular crosslinking
could be partially restored by cleaving the spacer
arm of DSP [Fig. 5(C,D)]. Similar intramolecular
crosslinked species were also observed in the case of
vinculin tail fragment (V884-1066), which likewise
decreased actin-bundling activity.22 This outcome
suggests that interdomain flexibility allows for an
open conformation is critical for actin-bundling
activity and is more important for bundling than for
binding to actin. Our results suggest that actin binding induces a conformational change in Ig3–4 that
favors both dimerization and bundling; therefore,
actin plays an active role alongside palladin in
PROTEIN SCIENCE VOL 24:70—80
77
transforming cytoskeletal organization. This active
role for actin in controlling bundling is a mechanism
common to structurally different actin bundling proteins, as previously identified in WLIM1 from Nicotiana tabacum, where actin promotes and stabilizes
the monomer to dimer transition. This dimerization
of WLIM1 then promotes actin crosslinking.36 Vinculin uses a similar mechanism, where actin inhibits
native dimerization of the tail fragment but exposes
a cryptic dimerization site that is essential for actin
bundling through conformational change in tail
domain.22
We further analyzed the actin-induced conformational change in Ig3–4 by limited proteolysis;
and, as seen with vinculin tail domain (Vt), conformational changes within the domain brought about
after actin binding resulted in an increase in protease susceptibility. Contrary to what was observed for
Vt, actin binding by Ig3–4 results in diminished protease reactivity. This suggests that an open conformation of Ig3–4 is induced or favored by actin
binding, which results in stronger Ig3–4 homodimerization
and
subsequently
decreases
the
susceptibility.
We demonstrate that the actin-binding and bundling domains of palladin remain monomeric in the
absence of actin, but oligomerize following incubation with actin. Our evidence that actin binding
decreases the susceptibility of palladin to proteases
supports the idea of actin-induced restrictions in
conformational flexibility of palladin. Interestingly,
we also observed that restricted domain movements
brought about by intramolecular crosslinking prevented actin binding and bundling by palladin.
Together our results provide a causal link between
actin-palladin interactions and oligomer formation.
Similar actin-induced protein oligomerization mechanisms have been reported for vinculin, vilin, and
WLIM1.22,36,37 Our results suggest that palladin
joins these structurally unrelated ABPs in a common
mechanism, whereby control of actin-bundling is
facilitated by actin itself. On the basis of these
results we put forth a model for palladin dimerization and bundling activity, where actin induces a
conformational change in palladin that gives rise to
a palladin structure that is more amenable to dimerization (Fig. 7). This conformational change in turn
promotes binding to multiple actin filaments and
subsequent crosslinking of these actin filaments into
tight bundles.
Materials and Methods
Materials
Muscle acetone powder for actin purification was purchased from Pel-Freez Biologicals (Rogers, AR). Crosslinkers DFDNB (1,5-difluoro-2,4-dinitrobenzene),
DSP or Lomant’s reagent [dithiobis(succinimidyl
78
PROTEINSCIENCE.ORG
propionate)], and BS3 [bis(sulfosuccinimidyl)suberate], and trypsin were purchased from Thermo
Fisher Scientific (Waltham, MA). Chymotrypsin was
obtained from Worthington Biochemical Corporation
(Lakewood, NJ). Polyclonal IgG antibodies against
palladin and alpha actin were purchased from Proteintech (Chicago, IL). Biotinylated anti-IgG secondary antibody and the immune detection system
R Elite ABC kits) were purchased
(VECTASTAINV
from Vector Laboratories (Burlingame, CA).
Expression and purification of recombinant
palladin protein domains
The coding sequences of Ig3, Ig4, and Ig3–4 domains
of palladin, previously cloned into pMAL-c2x,24 were
subcloned into the pTBSG vector containing an Nterminal hexa-histidine tag and TEV protease cleavage site for purification of recombinant proteins.38
pTBSG Ig3–4 was further modified by inserting the
MBP coding sequence between the histidine tag and
TEV protease site to avoid aggregation during
recombinant protein expression. All constructs were
verified by sequencing and were transformed into
BL21(DE3) derived T7 Express lysY competent
Escherichia coli cells (New England Biolabs; Ipswich, MA) for protein expression. Cell cultures were
grown at 37 C and induced by 0.5 mM IPTG at
OD600 of 0.7 and further grown overnight at 18 C.
Cell cultures were pelleted down and resuspended in
lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10
mM imidazole) followed by sonication and clearing
of cell lysate by centrifugation. Supernatant was
loaded onto a pre-equilibrated Ni-NTA column and
protein was purified according to the manufacturer’s
guidelines (Thermo Fisher Scientific, Waltham, MA).
The N-terminal histidine tag was cleaved by TEV
protease and further purified as previously
reported.24 All three palladin domains were finally
dialyzed in HBS buffer (20 mM HEPES, pH 7.4, 100
mM NaCl).
Chemical crosslinking
Chemical crosslinking was done on purified Ig3, Ig4,
and Ig3–4 proteins in the presence and absence of
F-actin. We have used three crosslinkers (DFDNB,
DSP, and BS3) that react with primary amines and
differ in their spacer arm length with 3.0 Å, 12.0 Å,
and 11.4 Å, respectively. The DSP crosslinker was
used for its DTT or TCEP cleavable disulfide bond in
the spacer arm. Stock solutions of 2 mM DFDNB,
DSP, and BS3 were prepared just before each experiment by dissolving the crosslinker in DMSO and/or
water, respectively. All crosslinkers were used at
final concentrations ranging from 15 to 100 mM and
crosslinking reaction times ranging from 10 to 40
min, then reaction was quenched by addition of
Laemmli buffer. G-actin for crosslinking experiments
was prepared from the purified actin stock made by
Actin-Induced Dimerization of Paladin
dialyzing G-actin in to buffer containing 2 mM
NaHCO3, pH 7.6, 0.2 mM CaCl2, 0.2 mM ATP, and
0.02% NaN3.
for 0 to 80 min. The reaction was quenched by addition of 1 mM PMSF. Samples were run on 12–15%
polyacrylamide gel.
Immunoblotting
Acknowledgments
Homo- and hetero-dimers of palladin and actinpalladin fragments were verified by immunoblotting.
Samples containing palladin and actin with and
without crosslinkers were run on SDS-PAGE in
duplicate and transferred on to PVDF membranes
(Millipore). Membranes were blocked at 4 C for 6 h
with 5% skim milk in TBS-T (0.05% Tween-20).
Blots were incubated overnight at 4 C with primary
antibody against palladin (1:1000 dilutions) or alpha
actin (1:1000 dilutions). Blots were washed with
TBS-T buffer and further incubated for 1 h at 25 C
with biotinylated anti-rabbit secondary antibody
(1:500 dilution) followed by incubation with Vectastain ABC solution for 30 min. For detection, 1 mg/
mL DAB was dissolved in 100 mM imidazole, pH
7.0, 2.5 mM CoCl2 and immediately before use
0.002% H2O2 was added. The reaction was stopped
by washing with water twice.
The authors thank Owen Nadeau at KUMC for his
assistance with pilot chemical crosslinking studies of
palladin, Philip Gao, and Anne Cooper in the
COBRE-PSF Protein Production Group at KU for
cloning of pTBSG vectors, and Isabel Hendry at
WSU for help with Western Blots.
Actin co-sedimentation assay in presence
of crosslinkers
Actin co-sedimentation assays in the presence of
crosslinker were carried out by incubating both the
crosslinker and protein at various concentrations,
and followed by addition of F-actin in buffer containing 10 mM Hepes, pH 7.5, 100 mM KCl, 2 mM
MgCl2. Samples were incubated for 60 min at room
temperature then reaction was quenched by addition
of 1M Tris, pH 7.5 and centrifuged at 100,000g for
30 min. Supernatants were removed carefully followed by pellet resuspension in 2X SDS buffer. Samples were run on a 12 or 15% polyacrylamide gel
according to the requirements of the experiment.
Gels were stained in PageBlue protein staining solution (Thermo-Fisher) and analyzed by using
ImageJ.39 Bundling of F-actin by Ig3–4 was carried
out in the presence of crosslinker as before except
with an additional step involving centrifugation of
samples at low speed (5,000g) to pellet high molecular weight bundled actin before high speed
centrifugation.
Limited proteolysis
Ig3–4 was incubated in presence of F-actin in 20
mM Hepes, 100 mM NaCl buffer at room temperature for 60 min, and then centrifuged at 150,000g
for 30 min. The supernatant containing unbound
Ig3–4 was separated and the pellet containing actin
bound Ig3–4 was then re suspended in 20 mM
Hepes, 100 mM NaCl. Both the unbound and bound
Ig3–4 were then digested by the addition of either
chymotrypsin (1:500) or trypsin (1:1000) to each
sample, followed by incubation at room temperature
Vattepu et al.
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Actin-Induced Dimerization of Paladin