Interaction of the N- and C-terminal Domains of Vinculin
CHARACTERIZATION AND MAPPING STUDIES*
Gregory J.
Miller,
Stanley D.
Dunn, and
Eric H.
Ball
From the Department of Biochemistry, University of Western Ontario,
London, Ontario, N6A 5C1, Canada
Received for publication, September 21, 2000, and in revised form, November 26, 2000
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ABSTRACT |
The vinculin head to tail intramolecular
self-association controls its binding sites for other components of
focal adhesions. To study this interaction, the head and tail domains
were expressed, purified, and assayed for various characteristics of
complex formation. Analytical centrifugation demonstrated a strong
interaction in solution and formation of a complex more asymmetric than
either of the individual domains. A survey of binding conditions using a solid-phase binding assay revealed characteristics of both
electrostatic and hydrophobic forces involved in the binding. In
addition, circular dichroism of the individual domains and the complex
demonstrated that conformational changes likely occur in both domains
during association. The interaction sites were more closely mapped on the protein sequence by deletion mutagenesis. Amino acids 181-226, a
basic region within the acidic head domain, were identified as a
binding site for the vinculin tail, and residues 1009-1066 were
identified as sufficient for binding the head. Moreover, mutation of an
acidic patch in the tail (residues 1013-1015) almost completely
eliminated its ability to interact with the head domain further
supporting the significance of ionic interactions in the binding. Our
data indicate that the interaction between the head and tail domains of
vinculin occurs through oppositely charged contact sites and results in
conformational changes in both domains.
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INTRODUCTION |
Focal adhesions are dynamic complexes of structural and signaling
proteins located at sites of very close contact between the
extracellular matrix and the plasma membrane. These complexes function to link filamentous actin to the extracellular matrix via
integrin receptors and a number of cytoplasmic proteins. Vinculin, a
soluble 116-kDa protein, is a component of focal adhesions and also
plays a role in cadherin-mediated cell-cell adhesion (1). Although the
specific role of vinculin within focal adhesions is unknown, genetic
approaches have been used to demonstrate the importance of vinculin in
both cell culture and animal model systems (2, 3).
Vinculin possesses no known enzymatic activity and is believed to play
its crucial role through its ability to associate with a large number
of other cytoskeletal and signaling proteins. Vinculin has been shown
to associate with talin,
-actinin, paxillin, F-actin, and members of
the VASP/Ena and ponsin/ArgBP52/vinexin families (4-7). Interactions
such as these may play an important role in vinculin function as its
association with focal adhesions has been shown to rely on the presence
of protein components in these structures (8). By binding to both
structural and signaling proteins and bringing them into proximity,
vinculin may act as an adaptor protein and may play an important
structural role through cross-linking actin filaments.
Structurally, vinculin is composed of two domains: a large 90-kDa
globular head and a 30-kDa elongated tail connected by a proline-rich
hinge region. A strong interaction (Kd ~50 nM) between the vinculin head and tail domains has been
observed (9), and inactivation of binding sites for other proteins on both the head and the tail domains has been demonstrated to occur during this self-association. For example, both the binding of the head
domain to talin and the binding of the tail to actin filaments are
inhibited by the head-tail interaction (10, 11). These observations
have led to a model for the vinculin activation in which cytosolic
vinculin exists in an inactive, "closed" state, and factors that
inhibit the head-tail binding induce vinculin to adopt an active,
"open" conformation allowing its recruitment into focal adhesions.
The binding sites involved in this intramolecular interaction have been
investigated previously using deletion mutagenesis. The deletion of
residues 167-207 resulted in an inability of the head domain to bind
the tail in vitro (12) suggesting that this small region
either was structurally important for activity or contained the binding
site. C-terminal truncation mutants of the vinculin head domain have
been used to localize the tail-binding site to the N-terminal 258 amino
acids (12), but no attempts have been made to more clearly define the
boundaries of this site.
Similar strategies have been used to identify the head-binding site
within the vinculin tail domain. C-terminal deletions of the vinculin
tail were used to define the C-terminal boundary of the site to between
residues 1028 and 1036 (9), but the extent of the binding region is not
clear. Interestingly, the C-terminal region of the vinculin tail has
also been implicated in binding to actin, phospholipids, and paxillin
and is one of two sites responsible for focal adhesion targeting of
vinculin (13-16). Bakolitsa et al. (17) recently solved
the crystal structure of the isolated tail domain and demonstrated that
it adopts an apolipoprotein E-like four-helix bundle with a C-terminal
arm. Many of the tail-binding sites lie within helix 5 and the terminal arm of the vinculin tail.
To investigate the vinculin intramolecular association, we have
separately expressed recombinant polypeptides of the vinculin tail
domain and the N-terminal 266 amino acids of the vinculin head region.
We have used these constructs to examine their shapes individually and
in a complex by analytical centrifugation and circular dichroism
spectroscopy. Further, we developed a solid-phase binding assay to
characterize their interaction quantitatively and map regions of the
polypeptides sufficient to support the interaction. In this paper, we
demonstrate the head-tail binding to be strongly ionic in character and
propose that it is the result of the interaction between two oppositely
charged faces of amphiphilic helices: a basic region (residues
182-226) within the acidic head domain and an acidic region (residues
1009-1066) within the highly basic tail. In agreement with this, we
find that residues Asp-1013, Glu-1014, and Glu-1015 in the tail
domain are necessary for interaction with the vinculin head domain.
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EXPERIMENTAL PROCEDURES |
Recombinant Proteins--
Recombinant DNA techniques were from
Sambrook et al. (18). Vinculin domains were amplified from a
cDNA pool synthesized from the reverse transcription of total human
RNA. Initially, DNA encoding vinculin
residues 5-266 was amplified using primers 1 and 2 (Table
I). This
PCR1 product was cloned into
the XbaI and SalI sites of the pGEX-KG vector
(19). Subsequently, this plasmid was used as a template in a PCR
reaction using primers 3 and 2 to add the five N-terminal residues.
This product was cloned into the PCRBlunt (Invitrogen, Carlsbad, CA)
vector and subsequently into the NcoI and SalI
sites in the pGEX-KG vector creating construct pKG-V-(1-266).
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Table I
Primer sequences used for PCR amplification of vinculin deletions
Sequences corresponding to vinculin cDNA are underlined.
Non-underlined sequences are 5' restriction endonuclease sites.
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Truncations of pKG-V-(1-266) were created using the internal
HindIII and SacI restriction endonuclease sites.
Construct pKG-V-(1-86) was created by digestion of pKG-V-(1-266) with
HindIII and religation. pKG-V-(1-181) was created by
digestion with SacI and religation. N-terminal truncations
were constructed after digestion of pKG-V-(1-266) with either
HindIII or SacI, isolation of the cleaved
fragment, and re-insertion into the pGEX-KG vector to create
pKG-V-(87-266) and pKG-V-(182-266), respectively. pKG-V-(182-226)
was created by the digestion of pKG-V-(87-266) with HindIII
and religation.
To construct vectors to express regions of the vinculin C-terminal tail
domain, cDNA encoding amino acid residues 833-1066 was amplified
using primers 4 and 5 (Table I). This PCR product was digested using
XbaI and XhoI and cloned into pGEX-KG. This construct was amplified using primers 4 and 6 and cloned into the
XbaI and XhoI sites in pGEX-KG. This construct
was used for further PCR reactions using primers 6 and 7 and to clone
two additional regions of the vinculin tail domain using primers 8 and
4. These two PCR products were cloned into a TA vector
(Invitrogen) prior to cloning into the XbaI and
XhoI sites of pGEX-KG thereby creating constructs
pKG-V-(877-964) and pKG-V-(1009-1066).
Site-directed mutagenesis of residues Asp-1013, Glu-1014, and Glu-1015
was performed using a PCR protocol (20). The resulting PCR product was
cloned, sequenced, and placed into pGEX-KG.
Constructs were expressed in BL21(DE3) or RR1 strains of
Escherichia coli. Cultures were shaken at 37 °C until an
A600 of 1.0-1.5 was reached when they were then
transferred to room temperature, and 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside was added.
Cultures were continuously shaken for an additional 6-8 h. Cells were
harvested, and the pellet was resuspended in 50 ml of TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) per liter of
bacterial culture. Protease inhibitors phenylmethylsulfonyl fluoride,
pepstatin A, leupeptin (Sigma), and EDTA were added prior to lysis of
cells by passage through a French press at 20,000 p.s.i. Cell debris
was removed through centrifugation for 20 min at 30,000 × g in a Beckman JA17 rotor.
Purification of proteins on glutathione-agarose beads (Sigma) was
performed in a manner similar to Ball et al. (21).
Modifications to this procedure included the use of a 2-ml
glutathione-agarose column rather than suspensions of
glutathione-agarose. Constructs V-(1-266) and V-(877-1066) were
cleaved from the GST moiety using 2 µg/ml thrombin (ICN, Irvine, CA)
at room temperature for 30 min and thereby eluted from the columns.
Other constructs were eluted from the columns using reduced glutathione.
Further purification of V-(1-266) and V-(877-1066) was performed on
ion-exchange columns. V-(1-266) was diluted 5-fold in TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.0) plus protease
inhibitors, applied to a DEAE-Sepharose column (Amersham Pharmacia
Biotech), and eluted with a linear 50-250 mM NaCl
gradient. V-(877-1066) was diluted 5-fold, applied to an S-Sepharose
column (Amersham Pharmacia Biotech), and eluted using a 50-200
mM linear NaCl gradient.
Analytical Ultracentrifugation--
Ultracentrifugation was
performed in a Beckman model XL-A analytical ultracentrifuge at
20 °C. Sample buffer contained 20 mM Tris-HCl, pH 7.5, 75 mM NaCl, 3 mM MgCl2, 1 mM EGTA, and 1 mM dithiothreitol. Scans were
taken at 280 nm. Sedimentation equilibrium experiments were performed
at 30,000 rpm with protein concentrations of 20, 30, and 40 µM. In experiments containing two components, each
component was at an equal concentration. Equilibrium was determined by
coincidence of scans taken >2 h apart. Sedimentation velocity
experiments were performed at 60,000 rpm with scans taken at 10-min
intervals. Data were analyzed by software provided by Beckman
Instruments. Partial specific volumes were calculated by
standard methods. In both one- and two-component experiments, data were
fitted to a model of a single ideal interacting species.
Protein Labeling and Solid-phase Binding Assay--
Proteins
were labeled using IODO-GEN (Pierce) as described (22); specific
activities of 1.5-4.0 × 106 cpm/µg of protein were
obtained. A solid-phase binding assay was performed on 96-well
polyvinyl chloride plates (Fisher). Unlabeled protein was coated on
wells at 15 µg/ml in TBS for 2 h, and then the wells were
blocked using 3% bovine serum albumin in TBS for 2 h. Labeled
protein was added at a minimum concentration of 10 nM in
100 µl. At this concentration, equilibrium was reached after 8 h
of incubation; therefore, all further assays were incubated for at
least this length of time. Binding was performed in 20 mM
MES, pH 6.8, 75 mM NaCl, 3 mM
MgCl2, 1 mM EGTA, and 0.1 mM 2-mercaptoethanol at 20 °C. After 8 h, unbound ligand was
aspirated and the wells were washed three times using binding buffer.
Wells were then separated, and bound radioactivity was measured in a Wallac model 1470 gamma counter. Nonspecific binding was determined for
each assay by determining the amount of radioactivity bound to the
wells in the presence of a 100-fold concentration of unlabeled protein
over added radioligand. The amount of protein adsorbed to the plate was
determined by coating wells with trace amounts of radiolabeled protein
in a total concentration of 15 µg/ml. After 2 h, unadsorbed
protein was removed, the wells were washed three times with TBS, and
radioactivity adsorbed to individual wells was measured.
When binding was performed at variable temperatures, wells containing
binding reactions were overlaid with mineral oil, and the plates were
wrapped in plastic film. Plates were placed in constant temperature
rooms or incubators. All binding data were analyzed using Prism
software (GraphPad Software, San Diego, CA). Reaction conditions within
each experiment were performed in triplicate. Data are presented as the
mean ± S.D.
Circular Dichroism Spectroscopy--
The far UV circular
dichroism spectra of vinculin domains V-(1-266) and V-(877-1066) was
determined using a Jasco-J810 spectropolarimeter. Each sample was
scanned over the range of 200-260 nm in a quartz cuvette with a path
length of 0.1 cm. Spectra were gathered using 2.5 µM
protein in 10 mM sodium phosphate, pH 6.8, with 50-150 mM NaCl. Spectra were analyzed to determine
-helix
content using the CDNN circular dichroism deconvolution program
(23).
Other Techniques--
Protein determination was performed by the
Bradford assay (Sigma) and SDS-polyacrylamide gels using the
Laemmli buffer system (24).
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RESULTS |
To begin our investigation of the interaction between the head and
tail domains of vinculin, we cloned and expressed regions of vinculin
previously demonstrated to interact (9). These constructs, V-(1-266)
and V-(877-1066), were purified in high yield (20-40 mg/liter culture) and in high purity (>95%) (Fig. 1) as assessed by scanning overloaded
lanes on an SDS-polyacrylamide gel.

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Fig. 1.
Vinculin constructs and purification of
V-(1-266) and V-(877-1066) domains. A, schematic of
the domain structure and deletion mutants used. The head, tail, and
proline-rich domain (PRD) are labeled. Numbers indicating
amino acid residues encoding each of the vinculin constructs used in
this work are listed. B, SDS-polyacrylamide gels of
purification steps in the isolation of V-(1-266) and V-(877-1066).
Lane 1, lysed cells expressing fusion protein. Lane
2, insoluble fraction of the cell lysate. Lane 3,
soluble fraction of the cell lysate. Lane 4, protein eluted
from the glutathione-agarose column using thrombin. Lane 5,
isolated protein after further purification on an ion-exchange column.
MW, molecular weight.
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Analytical centrifugation was used to measure the shape and interaction
of the domains in solution (Table II). Data in each case were analyzed
as ideal single component systems to determine whether the individual
domains self-associate over a range of concentrations. The data are
presented as a ratio of the observed molecular weight
(MO) to the theoretical molecular weight
(MT) (Table II). The observed molecular weights
for V-(1-266) and V-(877-1066) are 32,900 ± 750 and
22,900 ± 1000, respectively, which indicate that the domains act
as monomers over the concentration range tested (20-40
µM). To examine the interaction in solution, an equimolar
mixture of the two proteins was tested using sedimentation equilibrium.
Analysis of the data by fitting to a single ideal species gave a
molecular weight of 48,100 ± 1600, almost equal to the
expected weight of the heterodimer, 50,925. This result indicates that
the recombinant domains interact strongly to form a 1:1 complex. We
attempted to calculate the Kd by fitting the same
data to a dimerization model using the average of the monomeric
weights of each component (25), but the near totality of the
dimerization precluded determination of a dissociation constant. To
further analyze the overall conformation of the domains, sedimentation
velocity was used to determine hydrodynamic parameters. The
sedimentation coefficients were determined at 60,000 rpm at 20 °C
and were corrected to obtain s20,w, the
sedimentation coefficient in water at 20 °C. The
s20,w for V-(1-266) and V-(877-1066)
was determined to be 2.75 and 2.47, respectively, and was then used to
determine the frictional ratios of each domain (Table II). V-(1-266)
and V-(877-1066) have frictional ratios of 1.16 and 1.05, respectively, and thus appear to be globular. The complex of the two
domains had a frictional ratio of 1.22, implying that the complex is
more asymmetric than either of the two individual domains.
To examine the secondary structure of the expressed domains, circular
dichroism was used. V-(1-266) and V-(877-1066) were found to be 66 and 45%
-helical, respectively, by this method (Fig.
2). This fraction of
-helix calculated
from the spectra is low for V-(877-1066) in comparison with the
determined crystal structure of the vinculin tail, which shows this
domain to be ~60% helical (7). When V-(1-266) and V-(877-1066)
were mixed the resulting
-helical content was 46%. The complex is
clearly less
-helical than the theoretical average of the individual spectra (Fig. 2), indicating that conformational changes occur during
the association of the head with the tail domain. Given the magnitude
of the change, it is unlikely that a conformational change in either
one of the individual domains alone could account for the observed
spectra. For example, if a change in V-(877-1066) alone was
responsible for the observed change in the secondary structure, it
would require a 60% decrease of its
-helical content. Therefore, it
is likely that more moderate changes in the structure of both domains
occur during their association.

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Fig. 2.
Circular dichroism of V-(1-266) and
V-(877-1066) individually and as a complex. The spectrum of
V-(877-1066) (curve A), V-(1-266) + V-(877-1066)
(curve B), the calculated average of V-(1-266) and
V-(877-1066) (curve C), and V-(1-266)(curve D)
are shown.
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We used a solid-phase binding assay to confirm and extend the results
obtained from the analytical centrifugation experiments. A binding
isotherm demonstrated specific and saturable binding with a
Bmax of 7.2 ± 0.15 nM, which
indicates a stoichiometry of 1.1 ± 0.45 mol of V-(1-266)/mol of
V-(877-1066) (Fig. 3). The Kd of a single class of binding sites was determined to be 93 ± 10 nM (inset). The
binding was salt-sensitive with over 50% inhibition at 200 mM NaCl (Fig. 4) due to an
effect on binding affinity rather than on maximum binding capacity
(data not shown). This salt sensitivity was not due to alteration of the conformation of the recombinant domains as the circular dichroism spectra were unaffected over a concentration range of 50-150
mM NaCl (data not shown). The affinity of the interaction
was also affected by a temperature variation between 4 and 55 °C
(Fig. 5). Lower temperatures led to a
greater time to reach equilibrium (3 h at 45 °C versus
24 h at 4 °C) and resulted in an ~20-fold decrease in
affinity from 45 to 4 °C (Fig. 5A). Evidently the domains must be quite stable as binding was still observed at 55 °C.
The pH optimum of binding was 7.5 (Fig. 5B) with
strong declines below pH 7.0 and above pH 8.0. Variation of the
concentration of divalent cations (MgCl2,
CaCl2, MnCl2) and the addition of EDTA had no
effect on the interaction of V-(1-266) with V-(877-1066) (data not
shown). These results indicate that the reaction between V-(1-266) and
V-(877-1066) is mediated by both electrostatic and hydrophobic
forces.

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Fig. 3.
Binding of 125I-V-(1-2660) to
V-(877-1066) using a solid-phase binding assay. Each well of the
96-well plate was coated with 20 ng of V-(877-1066). The binding of
125I-V-(1-266) was measured in the presence of variable
amounts of unlabeled V-(1-266) (0-1 µM). Nonspecific
binding was subtracted from the amount bound to each well.
Inset, a Scatchard plot of data.
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Fig. 4.
Effect of NaCl on binding of V-(1-266) to
V-(877-1066). Binding of 125I-V-(1-266) to
V-(877-1066) immobilized on a 96-well assay plate in the presence of
0-500 mM sodium chloride was measured. Data are plotted as
the percentage of 125I-V-(1-266) bound to plate relative
to amount bound at 0 mM NaCl.
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Fig. 5.
Effect of temperature and pH on binding of
V-(1-266) to V-(877-1066). A, binding of
125I-V-(1-266) to V-(877-1066) immobilized on a 96-well
assay plate at temperatures ranging from 4 to 55 °C. Wells
containing binding reactions were overlaid with mineral oil, and assay
plates were wrapped in plastic film and incubated in constant
temperature incubators. B, binding of
125I-V-(1-266) to V-(877-1066) immobilized on a 96-well
assay plate was measured in 10 mM MES, 3 mM
MgCl2, and 1 mM EGTA with the pH ranging from
5.5 to 8.5.
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To better define the sites of interaction on the molecule, deletions
were made separately in both domains, and binding activities were
measured. Within residues 1-266, five deletion mutants were constructed and studied (Fig. 1). Unstable without the GST moiety, these deletions were used as fusions. The GST moiety had no effect on
binding of the domains when fused to either V-(1-266) or V-(877-1066) (not shown). The purified deletions of V-(1-266) were iodinated and
tested for their ability to bind V-(877-1066). Mutants containing residues 1-86 and 1-181 showed no ability to bind V-(877-1066), but
mutants GST-V-(87-226) and GST-V-(182-266) were able to bind to
V-(877-1066) with Kd values of 180 ± 20 nM and 770 ± 120 nM, respectively (Fig.
6). To further delineate this binding region, the sequence common to GST-V-(87-226) and GST-V-(182-266) was
expressed and tested for binding. This construct, GST-V-(182-226), bound to V-(877-1066) with a Kd of 280 ± 40 nM (Fig. 6). Thus, this segment is sufficient to support
binding to the tail although with a 3-fold decrease in affinity.

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Fig. 6.
Deletion mutants of V-(1-266) binding to
V-(877-1066). Binding of 125I-labeled GST-V-(1-86)
( ), GST-V-(1-181) ( ), GST-V-(182-266) ( ),
GST-V-(87-226) ( ), and GST-V-(182-226) ( ) to V-(877-1066) was
measured. Curves were produced by measuring the binding of
each 125I-labeled mutant in the presence of 0-5000
nM unlabeled mutant.
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Similarly, we created deletion mutants of the vinculin tail to help map
a minimal binding region for the head domain. Constructs GST-V-(877-964) and GST-V-(1009-1066) were designed to collectively include all known binding sites of ligands of the vinculin tail. These
proteins were expressed, purified, coated onto wells, and probed with
125I-V-(1-266) to determine whether the deletions
bind to the vinculin head (Fig.
7A). Of the two constructs,
only V-(1009-1066) supported binding to V-(1-266). This interaction
occurred with a Kd of 630 ± 50 nM
(Fig. 7A).

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Fig. 7.
Binding of mutants of V-(877-1066) to
V-(1-266). A, binding of 125I-V-(1-266)
to GST-V-(1009-1066) ( ), GST ( ), and
V-(877-1066/D1013A,E1014A,E1015A) ( ). Binding curves
were produced by binding 125I-V-(1-266) in the presence of
0-5000 nM V-(1-266). B, circular dichroism of
V-(877-1066) (curve A) and
V-(877-1066/D1013A,E1014A,E1015A) (curve B).
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The inhibition of binding between V-(1-266) and V-(877-1066) by salt
is indicative of an ionic interaction. Because the 182-226 sequence
was basic, we suspected that acidic residues within 1009-1036 would be
involved, in particular an acidic patch near residue 1013 seen in the
crystal structure. To test this possibility, site-directed mutagenesis
was used to change three acidic residues (Asp-1013, Glu-1014, and
Glu-1015) to alanine within V-(877-1066). The mutations had little
effect on the conformation of the domain as measured by circular
dichroism (Fig. 7B). The difference in the measured
-helical content for V-(877-1066) and
V-(877-1066/D1013A,E1014A,E1015A) was less than 1% helix, but the
mutant was inactive when assayed for its ability to bind to V-(1-266)
(Fig. 7A).
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DISCUSSION |
Regulation of protein function by intramolecular autoinhibition
has been recognized to occur within a diverse group of proteins (26).
In these proteins, activity is inhibited in the intramolecularly associated state, and separation of the self-associated domains via
binding of an activator, phosphorylation, or proteolysis restores activity. Expression of the separate interacting domains of vinculin facilitated characterization of the binding and better definition of
the regions of the polypeptides involved. Our measured
Kd of 93 ± 10 nM agrees with
earlier observations of a tight interaction (Kd
estimated at 50 nM) (9) between the separate domains, suggesting an extremely tight interaction within the whole molecule where the domains are physically connected. We have also found that the
interaction is sensitive to increases in ionic strength and
temperature. These data indicate that both electrostatic and hydrophobic forces play important roles in the interaction of the head
and tail domains. Furthermore, the observation that the association is
greatly decreased below pH 7 is in agreement with the observation that
the head and tail domains of vinculin remain tightly associated after
V8 protease cleavage within the proline hinge sequence but can be
separated at a low pH (27).
The observed effect of salt concentration on the interaction of the
head and tail domains is consistent with its observed effect on whole
vinculin. At low and physiological concentrations of salt, vinculin is
monomeric as observed using rotary shadowing and electron microscopy,
but at high salt concentration vinculin is seen as multimers of four to
six molecules (28, 29). Based on our data, high ionic strength might
lead to the dissociation of the head-tail interaction allowing
intermolecular interactions to occur. In addition, the
dissociation of the head and tail that is induced by certain lipids is
decreased at low ionic strength (30), possibly due to the higher
head-tail binding affinity.
As a first step toward identifying specific residues involved in the
interaction, deletions were used to further define the interacting
regions. In the N-terminal domain, we identified a small peptide
(residues 182-226) as a binding site for the vinculin tail. Although
this region has a somewhat reduced affinity in comparison with the
complete V-(1-266), it must contain the major binding determinants.
The location of the site between residues 182 and 226 likely explains
the inactivity of the 167-207 deletion mutant (12) due directly to
removal of interacting residues rather than structural change. This
region likely adopts an
-helical conformation as the homologous
region in the related protein,
-catenin, is also
-helical (31).
In addition, our circular dichroism measurements point to a high
-helical content (Fig. 2). It is interesting that construct
V-(1-266) has a theoretical pI of 5.11, whereas the tail-binding
region within it, residues 182-226, has a calculated pI of 8.16. Thus,
this binding site is a basic island exposed on the surface of an
-helix within the acidic head domain. This led us to look for an
oppositely charged region in the tail sequence as possible interaction
sites. It was unexpected that the shorter deletion mutant V-(182-226) rather than the larger V-(182-266) bound with a higher affinity, and
we suspect that the extra 40 residues either affect the folding of the
182-226 region or bind to it in competition with the vinculin tail.
There are a number of acidic residues in the 226-266 sequence (calculated pI is 4.8) that could interact with the basic V-(182-226) region. In the intact molecule, constraints of tertiary structure would
presumably prevent these effects.
Within the vinculin tail sequence, previous deletion analysis had
defined a C-terminal boundary to the head-binding site. Truncations of
the tail domain from 1066 back to residues 1043 or 1036 remain
competent to bind the head domain, but the tail domain lacking residues
1028-1066 is inactive (12). Our finding that residues 1009-1066 can
bind to the head limits the N-terminal boundary of the site to residue
1009, thus mapping the major site of contact to between residues 1009 and 1036. The reduced affinity of the V-(1009-1066) piece
(Kd 630 nM) versus the
V-(877-1066) (93 nM) for the head indicates that there are
sequences outside this area required for high affinity binding or
structure determination. It is clear, however, that this region
contains a site sufficient to support binding to the head. In the
recently determined crystal structure, this sequence forms a loop and
part of helix 5 at one end of the tail. Binding of the head to this
loop could result in an elongated complex consistent with the
ultracentrifugation data, which shows a higher degree of asymmetry than
either individual domain. Another notable feature of this sequence is
the preponderance of acidic residues; although the tail domain of
vinculin is basic overall with a theoretical pI of 9.15, the calculated
pI of residues 1009-1036 is 4.14. To verify our prediction that acidic
residues are critical for the interaction, we mutated three consecutive residues (Asp-1013, Glu-1014, and Glu-1015) to alanine and found, as
expected, a drastic decrease in binding affinity. Whereas the decreased
affinity of V-(1009-1066) for V-(1-266) indicates that other regions
of the tail contribute to the binding, the abrogation of binding as a
result of these mutations within V-(877-1066) shows that those
contributions alone cannot support the interaction. This clearly
demonstrates that the region 1009-1036 is the major contact site for
V-(1-266), and very likely one or more of the acidic residues mutated
are directly involved in the binding. Deletion experiments
demonstrating a requirement for residues 1028-1036 (12) are not
inconsistent with our results and indicate that 1028-1036 are also
necessary either to maintain the structure of the binding site or to
make significant contacts. Taken together, these results strongly
support ionic interactions between the oppositely charged sequences as
major binding determinants.
It is interesting to note that several other vinculin-binding sites are
dependent upon sequences near the 182-226 and 1009-1036 binding
sites. Talin has been demonstrated to bind the vinculin head (7) and is
dependent upon residues 167-207 (32). Further, a lipid binding domain
(residues 1016-1066), a paxillin-binding site (requiring residues
978-1066) (15), an actin filament-binding site, and an oligomerization
site (13) all have been demonstrated to be near the head-binding site
within the tail. The proximity of these sites suggests a direct
mechanism for interactions and regulation to occur. A simple model for
vinculin regulation postulates that phosphatidylinositol
4,5-bisphosphate "opens" vinculin by somehow causing
head-tail dissociation. A lipid-binding site near or coincident with
the head-binding site raises the possibility of direct competition.
Further experiments are needed to resolve the precise mechanism.
The conformational flexibility of vinculin is largely unexplored but
seems considerable. Our circular dichroism data show the first evidence
of conformational changes in the head domain, but there is supporting
evidence for distinct conformations of the tail domain in the presence
of actin or lipid (33). Evidence of conformational change in both of
these domains indicates that the head and tail domains may adopt
different conformations in the vinculin open and closed states
irrespective of ligand binding. The existence of multiple structures
suggests that a steric blocking model for vinculin regulation may be
too simplistic and that allosteric mechanisms may predominate. Thus,
vinculin is expected to show complex behavior in response to its
various binding partners and may function in both regulatory and
structural roles. The studies described here will provide a basis for
further investigation of this complicated protein.
 |
ACKNOWLEDGEMENTS |
We thank Aaron Siu and Sunil Shroff for the
construction of several of the deletion mutants used in this study and
Matt Revington for technical assistance with the analytical centrifuge
and the spectropolarimeter.
 |
FOOTNOTES |
*
This work was supported by Medical Research Council of
Canada Grant MT13349.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 519-661-3068;
Fax: 519-661-3175; E-mail: ehball@julian.uwo.ca.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M008646200
 |
ABBREVIATIONS |
The abbreviations used are:
PCR, polymerase
chain reaction;
TBS, Tris-buffered saline;
GST, glutathione
S-transferase;
MES, 4-morpholineethanesulfonic acid.
 |
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