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INTRODUCTION |
Fibronectin, a widely expressed extracellular matrix protein,
contains multiple modular regions that interact with various members of
the integrin family of heterodimeric cell-surface adhesion receptors.
The central cell binding domain, which is known to recognize
5
1,
v
3,
IIb
3 and at least eight other integrins, is particularly well characterized (for a review, see Ref. 1). This
region, spanning type III domains FIII8, FIII9, and FIII10, has become
the focus of intensive research as a model for the universal and
ubiquitous RGD-driven receptor-ligand interactions. The RGD tripeptide
motif, residing on a protruding, flexible loop within the FIII10 module
(2, 3), is essential for receptor binding and downstream cell adhesion
events such as pp125FAK-, Rho-, and
Rac-dependent signaling, cytoskeletal reorganization, and
acquisition of spread morphology (4-6). Although it is presently not
clear by which mechanism an RGD-containing ligand initiates a
conformational change in the integrin headpiece and transmittal of the
adhesive signal across the plasma membrane, the recent solution of the
crystal structure of integrin
v
3 in
complex with an isolated RGD peptide by Xiong et al. (7) is
an important first step toward elucidating the structural basis of the
FIII10-integrin interaction.
The FIII9 domain adjacent to FIII10 is known to act in synergy with the
latter in promoting integrin-mediated cell adhesion, drastically
increasing the activity of a single FIII10 domain but not having any
activity on its own (8). A number of sites within FIII9 have been
proposed to be critical for its synergistic effect. The most well
studied of these is the DRVPHSRN sequence or the so-called "synergy
site," the larger part of which is located on a surface loop of FIII9
lying approximately in plane with the RGD loop (3, 9) and thus being
potentially accessible for contact with the integrin dimer. Previous
studies have demonstrated that integrins binding to the FIII9-10
domain pair have a common requirement for the intact RGD motif, but
they display distinct preferences for individual residues within the
synergy site of FIII9 or may not need this site at all. For instance,
while high affinity
5
1 binding is most
sensitive to substitutions for Arg1379 (10), the most
critical residues for
IIb
3 appear to be
Asp1373 and Arg1374 (11), whereas
v
3 and
3
1
are indifferent to the presence of the FIII9 domain (11, 12). Other
amino acids in the vicinity of the classical synergy site (notably
Arg1369, Arg1371, Thr1385, and
Asn1386) have also been shown to contribute to the
synergistic activity of FIII9 (13), although no integrin specificity
has been determined for these residues. Taken together, these findings
support the notion that each distinct integrin-ligand interaction,
despite exhibiting some overall topological similarities, has its
idiosyncratic characteristics, which define the precise conditions for
an efficient and specific binding to the synergistic FIII9-10 module pair.
Biophysical studies have revealed that in comparison with FIII10, the
FIII9 domain is thermodynamically rather unstable (14) and displays
much slower folding kinetics (15). Coupling to the neighboring FIII
modules within fibronectin clearly plays an important role in
stabilizing FIII9, since it exhibits around 3-fold higher
conformational stability in module pairs FIII8-9 and FIII9-10 than on
its own (16), and altering its distance from FIII10 by introducing
interdomain linkers of varying length critically reduces its stability
(14). The relationship between the thermodynamic properties of FIII9
and its synergistic integrin-dependent biological activity,
however, remains little studied despite its likely important
implications for understanding the mechanism of integrin-ligand complex
formation. A previous investigation has shown that the decrease in
FIII9 stability due to the insertion of the above-mentioned linkers
between FIII9 and FIII10 coincides with the reduced adhesive activity
of the domain pair and impaired induction of intracellular signaling
(17). More recently, we were able to demonstrate a link between the
enhancement of biological function of the FIII9-10 pair by the FIII8
module and its positive influence on the structural stability of FIII9
(16). In the latter study we also probed the role of the FIII9 synergy
site in stabilizing the FIII8-9 pair within the FIII8-9-10 module
tandem, documenting a negative impact of perturbing the synergy
sequence in this three-domain context.
Here, we have specifically addressed the question whether, and to what
extent, do specific amino acid residues within the synergistic DRVPHSRN
loop contribute to the global stability of FIII9 and how this relates
to the functional performance of the FIII9-10 domain pair. We use a
combination of biophysical, biochemical, and biological methods to show
that the altered structural properties of various FIII9-10 synergy
site mutants are strongly linked to their integrin
5
1-dependent function.
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EXPERIMENTAL PROCEDURES |
Construction of Mutant pGEX2T-FIII9-10 Clones--
Cloning of
the wild type FIII9-10 domain pair into the pGEX2T vector (Amersham
Biosciences) has been described elsewhere (8). Amino acid
substitutions into the wild type FIII9-10 construct were introduced
following the QuikChangeTM protocol (Stratagene). The
following mutations were made to the synergistic DRVPHSRN sequence of
FIII9: Arg1379 to Ala (DRVPHSAN; mutant Ala1);
Asp1373-Arg1374 to Ala-Ala and
Ser1378-Arg1379-Asn1380 to
Ala-Ala-Ala (AAVPHAAA; mutant Ala5), as highlighted in Fig. 1. Creation of mutant Ala3, containing
the substitution of
Ser1378-Arg1379-Asn1380 with
Ala-Ala-Ala (DRVPHAAA), has been described previously (16). Constructs
harboring mutations in the synergy site were further mutated in FIII9
by substituting Leu1408 with Pro (mutants Ala1-L1408P,
Ala3-L1408P, and Ala5-L1408P). The latter substitution was also
introduced into the wild type FIII9-10 pair, resulting in mutant
L1408P (see Table I). The DNA sequence of all created mutants was
confirmed using Sanger DNA sequencing methodology (Department of
Biochemistry, University of Oxford).

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Fig. 1.
Ribbon diagram of the FIII9-10
domain pair. Atom co-ordinates were obtained from The
Protein Data Bank (24) (PDB ID: 1FNF) and imaged using the program
RasMol (www.umass.edu/microbio/rasmol). The substituted residues
(colored) are shown in space-fill mode and labeled accordingly.
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Expression and Purification of Recombinant FIII9-10
Proteins--
Wild type and mutant FIII9-10 constructs were expressed
in Escherichia coli as glutathione S-transferase
(GST)1 fusion proteins and
purified as described previously (8). For equilibrium chemical
denaturation studies, cleaved FIII proteins were obtained by thrombin
digest of the respective GST fusion proteins as described elsewhere
(16).
ELISA--
Integrin
5
1 used in
ELISA and surface plasmon resonance studies was purified from human
placenta as described previously (16). ELISAs with plate-bound
5
1 and soluble FIII9-10 ligands were
carried out in the presence of divalent cations as detailed earlier
(16). Assays were performed in duplicate, and background antibody
binding in the absence of ligand was subtracted from the readings.
Nonspecific binding of the GST fusion proteins to uncoated wells
blocked with bovine serum albumin was measured separately for
each ligand concentration point and subsequently subtracted from the
corresponding values for total binding. Dose-response data from the
assays were analyzed by non-linear regression using a sigmoidal curve
fit (Prism, GraphPad Software). Assay results are expressed as the
means of at least three independent experiments. Error bars represent
S.E. (dose-response curves) or 95% confidence intervals (bar graphs).
Surface Plasmon Resonance Studies--
Real-time biomolecular
interaction analysis was performed using a BIACORE 2000 instrument
(Biacore, Uppsala, Sweden). All experiments were performed at room
temperature using the standard HEPES-buffered saline as running buffer.
Briefly, 9000-16,000 resonance units anti-GST antibody
(Biacore) was immobilized on CM5 sensor chips (Biacore) according
to the manufacturer's instructions (BIAapplications Handbook,
Biacore). A volume of 30 µl of different GST-tagged FIII9-10
proteins diluted in HEPES-buffered saline were then injected at 5 µl/min to reach a constant level of binding. For kinetic
measurements, integrin
5
1 was injected
for 10 min at three different concentrations (80, 160, and 320 nM) at a constant flow rate of 5 µl/min. Both the
association and dissociation reactions were performed in Tris-buffered
saline (25 mM Tris, 150 mM NaCl, pH 7.4)
containing MnCl2, MgCl2, and CaCl2
at 2 mM each. The same injections of integrin were
performed on a GST protein used as a reference. The sensor surfaces
were regenerated with a short pulse of 10 mM glycine, pH
2.2. Data were analyzed by using the global fitting algorithm of the
BIAevaluation 3.0 software package (Biacore), using a simple 1:1
kinetic model. At least two independent experiments were performed for
each
5
1-FIII9-10 interaction. Error bars
represent S.D.
Cell Adhesion Assays--
Cell attachment and spreading assays
were performed with baby hamster kidney (BHK) fibroblasts as detailed
elsewhere (8). Cell attachment was quantified by staining adherent
cells with 0.1% crystal violet as described previously (8). The data
obtained are expressed as the means ± S.E. of at least three
independent experiments.
Equilibrium Chemical Denaturation Studies--
Equilibrium
unfolding experiments were carried out on recombinant GST-cleaved
FIII9-10 proteins, incubated in 0-4 M guanidinium hydrochloride (GdnHCl) in phosphate-buffered saline, as described previously (16). Measurements were performed at 25 °C on a Shimadzu RF5001PC spectrofluorimeter, using an excitation wavelength of 278 nm.
Fluorescence emitted was recorded at wavelengths ranging from 360 to
425 nm. Data from the experiments were fitted for a two-state unfolding
mechanism as described earlier (20). Error bars in the bar graphs
represent 95% confidence intervals.
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RESULTS |
A Leu1408 to Pro Substitution Increases the Affinity of
FIII9-10 Mutants to
5
1--
To evaluate
the effect of perturbing the synergy site of FIII9 on integrin
5
1 recognition, solid-phase receptor
binding assays were performed with wild type FIII9-10 ligand and
mutants Ala1, Ala3, and Ala5 (Fig. 2),
incorporating various substitutions within the DRVPHSRN sequence (see
Table I). The apparent
Kd of the interaction (ligand concentration at the
midpoint of the dose-response curves shown in Fig. 2a) was
observed to increase gradually as more substitutions were introduced,
rising from ~2 nM for the wild type to ~5
nM for mutants Ala1 and Ala3 and ~15 nM for
mutant Ala5 (Fig. 2b, closed bars on the
left-hand panel). This dependence of high affinity
5
1 binding on the intact synergy site was
in agreement with previous studies (16, 18) and further demonstrated
the differential and co-operative effect of individual synergistic
residues in securing efficient receptor recognition.

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Fig. 2.
Solid-phase binding of recombinant wild type
and mutant FIII9-10 proteins to integrin
5 1
as measured by ELISA and SPR. a, dose-response
curves for wild type FIII9-10 ( ) and mutants Ala1 ( ), Ala3
( ), and Ala5 ( ). Results are normalized and expressed as
percentages of maximum binding activity. b, comparison of
the apparent Kd values for wild type FIII9-10
(wt) and mutants Ala1 to Ala5 either without (closed
bars) or with (open bars) a Leu1408 to Pro
substitution, as derived from ELISAs (left-hand panel) and
SPR studies (right-hand panel). Note that lower apparent
Kd values reflect higher receptor affinity and vice
versa.
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The results obtained by ELISA were confirmed using surface plasmon
resonance (SPR), allowing the monitoring of real-time kinetic parameters of the ligand-integrin complex formation. The apparent Kd values for the
5
1-FIII9-10 interaction calculated by
this method showed a similar increase upon introduction of mutations
into the synergy loop of FIII9, being lowest for native FIII9-10
(~17 nM) and highest for mutant Ala5 (~63
nM) (Fig. 2b, closed bars on the
right-hand panel). This difference stemmed from both the
slower association (on-rate) and faster dissociation (off-rate) of the
integrin from the mutated FIII9-10 mutants, as compared with the wild
type (data not shown). The discrepancy between the relative
Kd values from these experiments and the ELISA data
(the affinity of the least active mutant versus wild type
decreasing ~4- and ~8-fold, respectively) is likely due to the
different assay formats used. SPR analysis was conducted with captured
fibronectin ligands and soluble integrin, whereas immobilized integrin
and soluble fibronectin fragments were used for ELISAs. Also, in SPR
studies the integrin binds to the captured ligand in constant flow
conditions, whereas the ELISAs are conducted in the absence of flow,
which may have contributed to the variance of the relative affinities
of individual FIII9-10 ligands. Nevertheless, both techniques show a
clear dependence of binding affinity on the native structure of the
synergy loop and further suggest that this synergy requirement is
maintained regardless of the particular method of presentation of the
FIII9-10 ligand to the integrin.
Earlier studies in our laboratory have shown that a single
Leu1408 to Pro mutation within the FIII9-10 domain pair is
able to confer a significant increase in structural stability to the
native FIII9 domain maintaining an intact synergy loop (19). We
therefore sought to assess the functional consequences of this
substitution in the context of wild type FIII9-10 as well as synergy
site mutants Ala1, Ala3, and Ala5 with relatively poor integrin binding
properties. ELISAs performed with the various FIII9-10 mutants and
immobilized
5
1 (Fig. 2b,
open bars) revealed that mutant L1408P bound to the integrin
with ~3-fold higher affinity than the wild type (apparent Kd
0.7 nM), and the same
substitution in mutant Ala5-L1408P resulted in a similar ~3-fold
enhancement of function over Ala5. A modest improvement in
5
1 binding capacity was also seen with mutants Ala1-L1408P and Ala3-L1408P in comparison with mutants Ala1 and
Ala3, respectively.
Leu1408 to Pro Mutation Restores Cell Adhesion Activity
of FIII9-10 Synergy Site Mutants--
Having determined the
solid-phase integrin binding properties of the aforementioned FIII9-10
mutants, we proceeded to test the ability of these fibronectin
fragments to support integrin-mediated adhesion of BHK fibroblasts,
which express integrins
5
1 and
v
3 (17), as a physiologically more
relevant measure of biological activity (Fig.
3). Quantification of cell attachment to
protein-coated surfaces (Fig. 3a) revealed that wild type
FIII9-10 and mutant Ala1 were significantly better adhesive substrates
for BHK cells than mutants Ala3 and Ala5, the latter retaining only
~50 and ~30% of the wild type activity, respectively, at a coating
concentration of 1 µM. Cell spreading, a secondary event
in the process of cell adhesion, was even more sensitive to
perturbations in the synergy loop; the level of cell spreading at the
same coating concentration dropped to ~70 and ~40% of wild type
FIII9-10 activity for Ala1 and Ala3, respectively, while the response
for Ala5 was barely ~10% of that of the wild type (Fig.
3b).

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Fig. 3.
Quantification of cell adhesion promoting
ability of FIII9-10 synergy site mutants. Attachment
(a and c) and spreading (b and
d) of BHK cells on surfaces coated with FIII9-10 ( ),
Ala1 ( ), Ala3 ( ), Ala5 ( ), L1408P ( ), Ala1-L1408P ( ),
Ala3-L1408P ( ) or Ala5-L1408P ( ). Results are expressed in
arbitrary units (a and c) or as percentages of
cells spread (b and d). For clarity, separate
graphs are shown in c and d for each mutant pair
without (closed symbols) or with (open symbols) a
Leu1408 to Pro substitution.
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The FIII9-10 mutants carrying an L1408P substitution were tested in
these assays to determine whether this mutation was able to recover the
adhesion promoting capability lost in the FIII9-10 synergy site
mutants. The adhesive properties of mutant L1408P were enhanced in
comparison with the native FIII9-10 at lower coating concentrations,
confirming previous findings. Cell attachment on mutants Ala3-L1408P
and Ala5-L1408P was restored to wild type levels, being ~2- and
~4-fold more efficient compared with Ala3 and Ala5, respectively, at
1 µM coating concentration (Fig. 3c). Attachment to Ala1-L1408P, however, was reduced in comparison with
Ala1. The spreading of BHK cells was likewise affected by the L1408P
substitution (Fig. 3d). The activity of mutants Ala3-L1408P and Ala5-L1408P was significantly increased compared with Ala3 and
Ala5 (~2- and ~8-fold, respectively, at 1 µM coating
concentration). Cell spreading on mutant Ala1-L1408P remained at the
same level as that supported by mutant Ala1.
Leu1408 to Pro Substitution Rescues Reduced FIII9
Stability in Synergy Site Mutants--
Previous experiments with
longer fibronectin fragments spanning domains FIII8 through FIII10 had
indicated a role for the FIII9 synergistic sequence in contributing to
the structural stability of the FIII8-9 pair (16). In this study, we
explored further the correlation between the differential functional
activity of FIII9-10 constructs harboring various mutations in the
synergy site and their thermodynamic properties. Data from equilibrium unfolding experiments performed with FIII9-10 mutants using GdnHCl as
a denaturant are presented in Fig. 4 and
Table II. Global unfolding of the
FIII9-10 domain pair is known to occur by a well defined two-step
transition allowing for separate analysis of unfolding parameters for
the individual FIII9 and FIII10 domains (14, 16). Since the stability
of FIII10 does not depend significantly on the presence of FIII9 (16),
only the first step of the denaturation curve, corresponding to the
unfolding of FIII9 (14), was of interest here and is shown in isolation
for clarity. The analysis used assumes a two-state domain unfolding
mechanism as described by Pace and Scholtz (20). While FIII9 in mutant
Ala1 displayed roughly the same unfolding properties as in wild type
FIII9-10, the FIII9 module in mutant Ala3 was destabilised, an effect
that was more pronounced for mutant Ala5 (Fig. 4a). The
concentration of denaturant leading to 50% domain unfolding
([GdnHCl]1/2) dropped from 1.9 M for wild type
FIII9 to 1.5 M for Ala3 and to 1.2 M for Ala5
(Fig. 4b, closed bars), and the respective
negative shifts in the free energy of unfolding
(
G(H2O)) relative to the
wild type increased from
1.0 kcal mol
1 (Ala3) to
1.7
kcal mol
1 (Ala5) (Table II). These results thus
demonstrate that the thermodynamic stability of FIII9 within the
FIII9-10 pair is sensitive to specific amino acid substitutions within
the synergy loop. When unfolding curves for FIII9 in mutants
incorporating the Leu1408 to Pro substitution were
assessed, a substantial shift to the right was seen for all
Pro1408 mutants as compared with their Leu1408
counterparts (Fig. 4c). The corresponding increase in
conformational stability of FIII9 as reflected by the respective
[GdnHCl]1/2 and
G(H2O) values
was ~1.5-fold for all Pro/Leu mutant pairs (Fig. 4b,
open bars, and Table II).

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Fig. 4.
Comparison of thermodynamic stability
parameters of related FIII9-10 species. Equilibrium denaturation
of FIII9 versus GdnHCl concentration in wild type and mutant
FIII9-10 proteins (a and c). The symbols and
graph display in c are the same as in Fig. 3. b,
comparison of [GdnHCl]1/2 values for the unfolding of FIII9
in FIII9-10 proteins without (closed bars) or with
(open bars) a Leu1408 to Pro substitution.
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Table II
Equilibrium unfolding parameters for the first denaturation step of the
wild type and mutant FIII9-10 domain pairs
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Finally, the observed differential thermodynamic stability of the
FIII9-10 mutants was compared with their functional activity by
performing correlation analysis. Mutant Ala1-L1408P was excluded from
linear regression calculations due to its anomalously high stability/activity ratio. The results of the analysis, plotted in Fig.
5, reveal a strong correlation between
the structural stability of the FIII9-10 proteins and mean biological
activity (Pearson correlation coefficient r = 0.92;
p = 0.004).

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Fig. 5.
Correlation between conformational stability
and biological activity of FIII9-10 mutants. The values of
[GdnHCl]1/2 for the unfolding of FIII9 in Ala5, Ala3, wild
type FIII9-10, Ala5-L1408P, Ala1, Ala3-L1408P, and L1408P (symbols
from left to right), plotted against their
respective mean biological activity values, are shown. The latter was
defined for each protein as the arithmetic mean of normalized relative
values for ELISA-derived apparent Kd, cell
attachment at 1 µM coating concentration, and cell
spreading at the same concentration and expressed as percentages of
maximum biological activity attained. The line represents
the results of linear regression analysis
(r2 = 0.84), indicating a good correlation
between the data sets.
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 |
DISCUSSION |
The current study was aimed toward elucidating the structural
basis for the synergistic biological activity of FIII9 in the FIII9-10
module pair of fibronectin. By the use of a range of methods, we have
established a distinct role for the DRVPHSRN synergy sequence within
the FIII9 domain in maintaining the overall conformational stability of
FIII9. Our findings demonstrate that: (i) perturbing the native
configuration of the FIII9 synergy site, required for efficient
integrin
5
1 binding and cell adhesion, leads to impaired domain stability; (ii) the Leu1408 to Pro
mutation can compensate for loss of biological activity of the synergy
site mutants; and (iii) a strong correlation exists between the global
structural integrity and synergistic function of the FIII9-10 pair.
We have used alanine scanning mutagenesis within the synergy site to
establish the structural importance of residues previously reported to
play a key role in integrin-dependent adhesive processes (10, 11, 13). In our experiments, the reduction in biological activity
for a single Arg1379 mutant (Ala1) is very similar to that
observed recently by Redick et al. (13). Further loss of
function upon mutating both the N- and C-terminal portions of the
DRVPHSRN sequence (in Ala5) is also in good general agreement with the
latter report. Further substitutions of amino acids outside the
DRVPHSRN site have been shown to have an effect in combination with
Arg1379 in FIII7-10 fragments (13), although it is not
clear to what extent these substitutions affect local or global
foldedness. While confirming earlier findings, we show here that the
perturbations introduced in the FIII9-10 mutants not only affect the
functionality of the synergy site but also have a strong negative
effect on the thermodynamic stability of the FIII9 domain. Equilibrium
denaturation experiments reveal that even though the synergy site
resides on a solvent-exposed surface loop protruding from the main
domain core, it contributes significantly to the overall foldedness of FIII9, as synergy site mutants carrying multiple substitutions unfold
at much lower denaturant concentrations than the wild type. The fact
that this effect is not observable for the single Arg1379
mutant points to the involvement of more extensive interactions between
the core and the synergy loop in conferring stability to FIII9.
The structure-function correlation seen with the synergy site mutants
is further reinforced by the finding that a single independent substituted residue (Pro for Leu1408) on the other side of
the FIII9 module (shown in Fig. 1), conceivably unable to make a direct
contact with either the synergy loop itself or the integrin receptor,
essentially reverses the effect of inactivating the synergy site both
in terms of biological function and structural characteristics of the
FIII9-10 pair. Such indirect influence on integrin
5
1-dependent activities is
therefore most likely exerted via global stabilization of the FIII9 module.
In a previous report, we studied the influence of the FIII8 module
within the FIII8-9-10 domain tandem upon the stability of FIII9,
showing a causal link between FIII8-induced enhancement of synergistic
biological activity and restoration of structural integrity of an
FIII9-10 pair containing a dysfunctional synergy site (16). On the
basis of these findings, we suggested that the requirement for the
DRVPHSRN sequence may only be critical for integrin binding in the case
of an isolated FIII9-10 pair. However, here we show that a
Leu1408 to Pro substitution within FIII9-10 achieves a
similar effect without the need for an additional N-terminal domain.
Taken together, these two studies imply that any sufficiently strong
influence producing an increase in the global stability of FIII9, be it a single critical amino acid substitution or a whole additional domain,
may overcome the requirement for an intact synergy site in securing
efficient integrin recognition. This kind of
conformation-dependent receptor competency appears to play
an important role in the regulation of adhesion-related cellular
responses. The latter notion may be relevant in the light of the
evidence that some integrins appear to modulate synergy site binding as
a function of their activation state. For instance,
5
1 may lose its dependence on the synergy sequence in the presence of stimulatory antibodies or Mn2+
ions (21), while
4
1 and
4
7 only recognize FIII9 when
constitutively activated (22). Thus the requirement for the synergy
site may to a large extent depend on the specific overall conformation of both the fibronectin ligand and the integrin receptor.
Earlier studies utilizing synergy site-derived peptides in isolation
(11) or in the context of a foreign FIII scaffold (10) have shown that
mutations in the DRVPHSRN sequence can affect integrin binding and cell
adhesion even in the absence of a native FIII9 fold. This suggests that
while maintaining conformational stability of FIII9 may be an important
function of the synergy loop, it is likely that this is not the only
mechanism whereby this region fulfils its synergistic potential and
that, as proposed by a number of previous reports, direct and specific
molecular interactions between residues in the synergy site and the
ligand binding pocket of the receptor are also crucial for
biological activity. As suggested previously (16), full uncoupling of
these two mechanisms of action may not be possible, and both need to be
taken into account when modeling the establishment and maintenance of
fibronectin-integrin interactions.
The specific stability parameters of the FIII9-10 domain pair appear
to depend upon the particular conditions in which domain unfolding is
evoked. For instance, while chemical denaturation unfolding experiments
employed in this and other studies show that the FIII9 module is
thermodynamically much less stable than FIII10, steered molecular
dynamics simulations using forced mechanical stretching of the module
pair predict that FIII9 may be mechanically more stable than FIII10
(23). The apparent contradiction between the two data sets may be
explained by consideration of the fact that forced unfolding is a
kinetic process that queries the height of energetic barriers
separating different folded states rather than the equilibrium energies
measured by chemical unfolding. Given that the forced unfolding method
mimicks the physiologically highly relevant stretching of the
fibronectin molecule under tension and that the FIII9 domain responds
differently in different unfolding scenarios, it would be interesting
to investigate how the synergy site mutants employed in the current
study would affect the mechanical (as opposed to thermodynamic)
stability of FIII9 and which specific intradomain interactions are most
vulnerable to disruption by stretching.
In conclusion, this report provides strong evidence in support of the
hypothesis that domain stability is an important determinant of
integrin
5
1-dependent
synergistic biological potential of the FIII9 module. Our observation
that relatively minor amino acid substitutions produce unexpectedly
large effects in the thermodynamic stability of FIII9 thus highlights
the requirement for careful structural evaluation of modular proteins
participating in similar receptor-ligand interactions.