From the Departments of Biochemistry and
¶ Microbiology and Immunology, Wake Forest University School of
Medicine, Winston-Salem, North Carolina 27157, the
Unita
Operativa of Biologia Strutturale, Istituto Nazionale per la Ricerca
sul Cancro (IST), c/o CBA, Genova, Italy I-16132, and the
** Department of Cell and Developmental Biology,
University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, August 29, 2002, and in revised form, November 5, 2002
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ABSTRACT |
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Integrin Integrins are a widely distributed family of heterodimeric
transmembrane receptors that anchor cells to extracellular matrix proteins and mediate two-way communication between the exterior and
interior of a cell (1, 2). The Receptor occupancy then sends an "outside-in" signal, leading to
integrin clustering and downstream activation of kinases, especially
focal adhesion kinase, that stabilize the integrin-mediated links
between extracellular matrix proteins and actin filaments (15-17).
Clustering of bound receptors may facilitate these processes by
increasing the local concentration of integrin-associated proteins, especially those that bind to the cytoplasmic tail of Our understanding of the molecular basis for integrin activation has
been considerably enhanced by the recent publication of crystal
structures for the extracellular domain of the
Concerning the forces that drive integrin clustering, we and others
have demonstrated that the ability of RGDX peptides to block the
function of If hydrophobic effects are important in these interactions,
thermodynamic principles (32-34) and experience with other
self-assembling systems (35-37) predict that the extent of
Based on our experience (28, 31, 49) and that of others (50) with
biophysical characterizations of the Reagents--
Cyclo(S,S)-L-lysyl-L-tyrosyl-glycyl-L-cystinyl-L-homoarginyl-glycyl-L-aspartyl-L-tryptophanyl-L-prolyl-L-cystine
(cHArGD) and
cyclo(S,S)-L-lysyl-L-tyrosyl-glycyl-L-cystinyl-L-arginyl-glycyl-L-aspartyl-L-tryptophanyl-L-prolyl-L-cystine (cRGD) (31) were synthesized and purified by the Protein Analysis Core
Laboratory of the Comprehensive Cancer Center of Wake Forest University
(Winston-Salem, NC) using previously described protocols (56). Each
peptide was shown to have the correct amino acid sequence and the
correct molecular mass, using an Applied Biosystems 475 automated
peptide synthesizer and a Quattro II triple quadropole mass
spectrometer (Micromass, Inc., Beverly, MA), respectively. Eptifibatide,
(N6-(aminoiminomethyl)-N2-(3-mercapto-1-oxopropyl-L-lysylglycyl-L- Platelet Isolation and Characterization
Procedures--
Platelet-rich plasma and gel-filtered platelets were
isolated from blood obtained by venipuncture from healthy, adult,
volunteer donors as previously described (58). Platelet counts were
determined with a Coulter MDII Cell Counter (Beckman Instruments,
Miami, FL). Platelet Biophysical Measurements--
UV absorbance measurements were
performed as a function of wavelength on a Beckman diode array
spectrophotometer with
Fluorescence emission spectra were obtained over the same temperature
range with an Aminco-Bowman series 2 luminescence spectrometer (SLM-Aminco, Rochester, NY) with samples contained in thermostatted quartz microcuvettes (Hellma Cells, Inc., White Plains, NY); an excitation wavelength at 278 nm was used (31). These data were also
corrected for background fluorescence; the signal from HSC-OG buffer
was found to vary by ±6% over the range 20-60 °C. The small fluorescence intensity from the tryptophan-containing ligand-mimetic peptide cHArGD in HSC-OG exhibited an ~2-fold hyperbolic
decrease with increasing temperature, as expected for intrinsic
fluorescence measurements (60).
Static and dynamic light scattering measurements were performed in a
Brookhaven Instruments BI-2030 AT correlator operated in conjunction
with a BI-200 SM light scattering photometer/photon counting detector
and a Spectra Physics 127 He-Ne laser (28); samples in quartz
microcuvettes were contained in a thermostatted refractive index
matching vat at temperatures in the range 20-40 °C. For
translational diffusion coefficient determinations, each intensity-normalized photon count autocorrelation function obtained for
the
Data collected from buffered HSC-OG indicated that the octyl glucoside
micelle size distribution did not change appreciably from 20 to
40 °C in that the scattering intensity varied by ±13% and the mean
particle diameter changed by ±12%; no
temperature-dependent trends were observed in these data.
Our results obtained with 30 mM OG are consistent with the
report of Aoudia and Zana (40), who found that both critical micellar
concentration for octyl glucoside (23-25 mM) and
aggregation number (107-92) exhibited minimal temperature dependence
from 20 to 60 °C.
Sedimentation velocity and equilibrium measurements were performed in a
Beckman Optima XL-A analytical ultracentrifuge (Beckman) equipped with absorbance optics and an An60 Ti rotor (28, 31). By
always including HSC-OG buffer or buffer + ligand-mimetic peptide in
the reference compartment, the resultant optical signals were corrected
for any temperature-induced effects on the solvent, although data
previously described demonstrated these effects to be minimal.
Sedimentation velocity data obtained at 20 and 40 °C were analyzed
using both SVEDBERG (version 1.04) and DCDT+ (version 6.31) software
(J. Philo, Thousand Oaks, CA) to obtain the weight average
sedimentation coefficient (sw) and distribution of
sedimenting species, g(s*), respectively (62). All
sedimentation coefficients reported here have been corrected for
solvent density and viscosity to obtain
s20,w values.
The absorbance versus radial distance data obtained by
sedimentation equilibrium were analyzed by nonlinear regression with WinNONLIN3 (63) to obtain weight average molecular weights
(Mw) for the
Because depletion of larger species can also influence the resultant
Mw data, the quantity of absorbing material
present in cells containing
In contrast, Mw data obtained for the
ligand-bound integrins showed a somewhat less pronounced dependence on
rotor speed and temperature: for example, the ratio
Mw (6000 rpm)/Mw (8000 rpm) was 0.907 at 30 °C, 0.851 at 37 °C, and 0.765 at 40 °C
for the Transmission Electron
Microscopy--
Threading/Homology Modeling--
Models of the
The Biological Activity of Integrin Ligands
The biological activities of the
IIb
3
clusters on the platelet surface after binding adhesive proteins in a
process that regulates signal transduction. However, the intermolecular
forces driving integrin self-association are poorly understood. This
work provides new insights into integrin clustering mechanisms by
demonstrating how temperature and ligand binding interact to affect the
oligomeric state of
IIb
3. The ligand-free
receptor, solubilized in thermostable octyl glucoside micelles,
exhibited a cooperative transition at ~43 °C, monitored by changes
in intrinsic fluorescence and circular dichroism. Both signals changed
in a direction opposite to that for global unfolding, and both were
diminished upon binding the fibrinogen
-chain ligand-mimetic peptide
cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by
dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for
IIb
3 complexed with cHArGD, cRGD, or
eptifibatide yielded large, favorable entropy changes partly offset by
unfavorable enthalpy changes. Transmission electron microscopy showed
that ligand binding and 37 °C incubation enhanced assembly of
integrin dimers and larger oligomers linked by tail-to-tail contacts.
Interpretation of these images was aided by threading models for
IIb
3 protomers and dimers based on the
ectodomain structure of
v
3. We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIb
3
complex is the classic example of a regulated integrin, a receptor
whose affinity for adhesive macromolecules is modulated by changes in
conformation and clustering (3-5). The receptor is maintained in a
default inactive state on circulating human blood platelets, possibly by interactions between the short cytoplasmic domains of the
IIb and
3 subunits or with
integrin-associated proteins (6-9). According to this model, proteins
like fibrinogen or fibronectin are prevented from binding to
extracellular region of
IIb
3 until a
platelet stimulus, such as thrombin, binds to a G-protein-coupled
receptor (3, 10, 11). A rapid cascade of intracellular events releases an inhibitory lock, sending a signal some 15 nm outwards to the ectodomain of
IIb
3, where a rearrangement
of intersubunit contacts leads to an "open" receptor with a
functional binding site (12-14). This process of converting
IIb
3 to an active form is referred to as
"inside-out" signaling (3).
3
(4, 18, 19). A positive feedback mechanism may also be at work on the
cell surface, in that integrin oligomers could be especially efficient
at capturing multimeric adhesive proteins (15, 20).
v
3 integrin, in the absence and presence
of ligand (21, 22). However, many questions remain about the
mechanistic details of the biomechanical coupling between the distant
intra- and extracellular domains of
IIb
3.
Previous experiments have shown that truncating either the
IIb or
3 cytoplasmic regions yields a
receptor that is constitutively active for inside-out signaling,
indicating that communication between the cytoplasmic domains maintains
the inactive state of the integrin (23, 24). We have recently shown
that a truncation mutant, lacking the
IIb cytoplasmic
domain, underwent further activation in the presence of a high affinity fibrinogen mimetic peptide resulting in the formation of oligomers (25). These observations reinforce the concept that clustering plays a
major role in the regulation of integrin affinity through outside-in
signaling (13, 15, 26).
IIb
3 (27) as well as to
perturb the conformation of its ectodomain and to promote "tail to
tail" oligomerization all increased with the hydrophobicity of the
residue in the X-position (28, 29). Likewise, we found that
eptifibatide, a cyclized fibrinogen-mimetic peptide with a tryptophan
residue in its integrin-targeting sequence (30), was especially
effective at promoting
IIb
3 oligomerization, as monitored by sedimentation equilibrium and electron
microscopy (31). These observations have led to the concept that
nonpolar receptor-ligand interactions contribute to both
integrin activation and self-association (28, 31). This article tests
that hypothesis by investigating the effects of both temperature and
ligand binding on
IIb
3
structure/stability.
IIb
3 oligomerization should increase with
increasing temperature. However, extracting thermodynamic data on a
transmembrane protein such as the
IIb
3 integrin requires pure protein isolated in a thermally stable environment amenable to spectroscopic studies. The neutral,
nondenaturing detergent octyl glucoside
(OG)1 (38, 39) is especially
well suited for biophysical characterizations of integral membrane
proteins because it exhibits a critical micellar concentration
and aggregation number that are nearly invariant from 20 to 60 °C
(40). Octyl glucoside has been used to study the structure and dynamics
of bacteriorhodopsin (41), mammalian rhodopsin (42), OmpF porin from
Escherichia coli (43), and the E. coli outer
membrane ferrichrome transporter FhuA (44). Extensive biophysical
characterizations have shown that octyl glucoside, even at a
concentration well above its critical micellar concentration, does not
perturb the quaternary structure of the soluble lens protein
-crystallin (45, 46). Furthermore, oligomerization studies of
subunits B777 (47) and B820 (35) of the light-harvesting complex
from Rhodobacter sphearoides have been performed in octyl glucoside micelles.
IIb
3
integrin in octyl glucoside, we recognize the limitations of
extrapolation from a detergent-solubilized protein to the physiological
situation. However, the responses of solubilized integrins to ligand
binding, such as conformational changes and oligomerization (28, 31), have been remarkably similar to the effects seen in cellular membranes (20, 51). In addition, Litvinov et al. (52) recently
demonstrated that both purified
IIb
3 and
IIb
3 on the platelet surface displayed comparable force histograms for fibrinogen binding. These observations probably reflect the fact that the ectodomain of
IIb
3 contains >90% of the integrin
residues and only one narrow nonpolar segment on each subunit traverses
the plasma membrane (28). Thus the model of a circumferential
transmembrane belt comprised of a limited number of protein-bound
detergent (43, 53) or lipid molecules (54, 55) established from high
resolution crystal structures of integral membrane proteins supports
the validity of the studies of
IIb
3 in
octyl glucoside micelles described here.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aspartyl-L-tryptophanyl-L-prolyl-cysteinamide, cyclic (1-6)-disulfide), was kindly provided by COR Therapeutics (San
Francisco, CA) where it has been developed as a pharmaceutical, Integrilin (57). Peptide concentrations were determined by quantitative amino acid composition analyses as previously described (31, 56).
Highly purified human fibrinogen (free of plasminogen and Factor XIII)
was purchased from American Diagnostica (Greenwich, CT) and highly
purified human
-thrombin was from Sigma.
IIb
3 occupancy was
determined with flow cytometric analysis using a Biocytex kit BX7001
(Marseilles, France) (25, 59). Platelet aggregation profiles were
obtained in a Chrono-Log model 500 aggregometer. Platelet adhesion to
fibrin was determined in a microtiter plate adhesion assay with
colorimetric read-out as previously described (25).
IIb
3 Purification--
Milligram
quantities of highly purified
IIb
3 were
isolated from outdated human blood platelets (American Red Cross, Triad Blood Center, Winston-Salem, NC) as previously described (28, 49).
Biophysical measurements were performed on peak integrin fractions
obtained by size exclusion chromatography at 4 °C on a 0.9 × 85-cm column of Sephacryl S-300 equilibrated in pH 7.4 buffer (HSC-OG)
containing 0.13 mol/liter NaCl, 0.01 mol/liter HEPES, 0.002 mol/liter
CaCl2, 3 × 10
8 mol/liter basic trypsin
inhibitor, 10
6 mol/liter leupeptin, 0.02% sodium azide,
and 0.03 mol/liter
n-octyl-
-D-glucopyranoside. Peak fractions
were then concentrated in an Amicon pressure concentrator with a PLHK
cellulose membrane, 100,000 Da retention limit (31).
IIb
3 samples
contained in 1-cm path length, 0.1-ml volume quartz cuvettes. Circular
dichroic spectroscopy was performed as a function of temperature from
20 to 60 °C in a Jasco model 720 spectropolarimeter (Japan
Spectroscopic Co., Tokyo) with samples in a thermostatted 0.05-cm
path-length cuvette; data are expressed as molar ellipticity, [
],
versus wavelength (25). In computing [
], each data set was corrected for the signal from the HSC-OG buffer or buffered octyl
glucoside containing the ligand-mimetic peptide cHArGD at the same
temperature. However, these background measurements changed by only
±5% over the range 20-60 °C.
IIb
3 complex was first corrected for
the contributions of octyl glucoside micelles. Following procedures
described in our earlier work (49), the autocorrelation function was
treated as an intensity-weighted sum of two exponential decay
components, one corresponding to the macromolecule and the other to the
faster moving detergent micelles. The contribution of the detergent was determined from separate measurements of buffered octyl glucoside, obtained under the same instrumental conditions. The resultant signal
was weighted by its fractional intensity, then subtracted from the
corresponding autocorrelation function for the integrin:detergent mixture to yield a corrected autocorrelation function. Each corrected autocorrelation function was then analyzed by the method of cumulants to obtain a z-average translational diffusion coefficient for the
IIb
3 complex. Size distribution
information was also obtained from these corrected autocorrelation
functions with the CONTIN algorithm (61). We note that CONTIN analysis
did not consistently identify a peak corresponding to the ~5-nm
diameter OG micelles in the uncorrected data, probably because we
worked under conditions where the detergent contributed less than 20%
of the total scattering intensity. All translational diffusion
coefficients reported here have been corrected for solvent viscosity to
obtain D20,w and Stokes radius
(Rs) values.
IIb
3
complex alone and in the presence of ligand-mimetic peptide.
Mw data were analyzed for their dependence on
rotor speed (at 6000 and 8000 rpm), a diagnostic for irreversible
aggregation (64). In the absence of ligands, data obtained with
IIb
3 yielded a ratio
Mw (6000 rpm)/Mw (8000 rpm) of 0.905 at 30 °C, 0.839 at 37 °C, and 0.737 at 40 °C. In
addition, global fits to data obtained at 37 and 40 °C yielded a
variance of ~3 × 10
3, a 5-fold decrease in
quality compared with fits obtained with data at lower temperatures.
These observations are indicative of thermal aggregation for the
ligand-free integrin (64).
IIb
3 at
equilibrium was computed and compared with that initially added. This
was done by numerical integration of the absorbance versus
radial distance profiles obtained at 30 and 40 °C, as well as the
initial, pre-equilibrium scans obtained at 3000 rpm. Whereas ~100%
recovery was achieved at 6000 rpm/30 °C, this parameter fell to
~68% at 8000 rpm/30 °C; because only absorbance values <3.0 were
included, there is some uncertainty associated with these estimates. In
addition, only data at absorbence <1.5 were included in the subsequent
analyses to ensure compliance with Beer's law, thus 47% of the
integrated area was used for fitting at 6000 rpm and 42% at 8000 rpm.
These effects became more pronounced at 40 °C where recoveries of 42 and 28% were obtained at 6000 and 8000 rpm, respectively; 33% of the
6000 rpm data and 25% of the 8000 rpm data were used for fitting.
IIb
3·cHArGD complex.
Global fits of data obtained in this temperature range, at two rotor
speeds, and receptor concentrations in the range 1.4 to 4.0 µM, yielded a variance of ~7 × 10
4,
indicating the data obtained for the ligand-bound integrin were more
consistent with oligomerization, rather than the aggregation behavior
observed for the free receptor. Sample depletion was also less
pronounced with the ligand-bound integrin samples, as recoveries of
94 ± 10% and fitted ranges of 42 ± 5% resulted (average of data at 6000 and 8000 rpm, 30 and 40 °C). Therefore, data
obtained with the integrin-ligand complexes were subjected to
additional analyses with WinNONLIN3 to obtain a set of
temperature-dependent self-association constants (64).
WinNONLIN3 has been provided by Dr. David Yphantis and the staff at the
National Analytical Ultracentrifugation Facility, Storrs, CT.
IIb
3 samples at ~1
mg/ml in HSC-OG (in the presence/absence of 20 µmol/liter cHArGD)
were incubated for 3 h at either 20 or 37 °C, then diluted to
~20-25 µg/ml in a buffer containing 0.05 mol/liter ammonium
formate at pH 7.4, 30 mmol/liter octyl glucoside, and 15% (v/v)
glycerol. Samples were then sprayed onto freshly cleaved mica and
shadowed with tungsten in a vacuum evaporator (Denton Vacuum Co.,
Cherry Hill, NJ) (65-67). These samples were examined in a
Philips 400 electron microscope (FEI Co., Hillsboro, OR) operating at
60 kV and a magnification of ×60,000. Counts of molecules with
different conformations or different amounts of oligomers were made
from prints of the micrographs, using images from many different areas
of several different preparations to get a random sample.
IIb
3 integrin were constructed using the
crystal structure of the extracellular domain of homologous receptor
v
3 (21) as a template. The resources at
SwissPdB Viewer (68) (www.expasy.ch.spdbv/) were used to thread the
sequence of the
IIb subunit (P08514) into the
three-dimensional structure of
v (Protein Database file
1JVA) (21). Initially, a model was developed using the "bent"
structure observed crystallographically (21). Because regions of the
3 subunit corresponding to epidermal growth
factor modules 1 and 2 were not well defined in the crystal structure (21), these segments were replaced with homology models (68)
developed from NMR structural data reported by Beglova et
al. (69). Subsequently, an "extended"
v
3 template was generated by rotating and
translating each subunit about its flexible pivot as outlined by Xiong
et al. (21). Thus, we obtained an extended
IIb model that exhibited a C
root mean square
deviation <1 Å with the
v structure; however, sequence
gaps and misalignments that could not be resolved precluded the
development of a complete homology model. Replacing the coordinates of
the
v with those of our threaded subunit yielded a model
for the extracellular domain of the
IIb
3 integrin.
IIb and
3 transmembrane domains were
modeled as helical segments, based on packing principles for
membrane-bound polypeptide chains. The helical segment for
IIb was extended toward its membrane-proximal region,
based on NMR data, which also defined the conformation of its
cytoplasmic domain (6). Secondary structure prediction algorithms identified a short helical segment in the cytoplasmic domain
of
3 (71), a concept reinforced by the NMR study of Ulmer et al. (72). These segments were joined to the
IIb
3 ectodomain to obtain a model of the
complete integrin. Integrin dimers were modeled in a tail to tail
configuration based on our electron microscopy observations (Refs. 28
and 31, and this work).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIb
3 ligands used in this study are
summarized in Table I. cHArGD and cRGD
have been described as high affinity analogs of the KQAGDV and RGD
integrin-recognition sites of fibrinogen, respectively (73);
eptifibatide is an FDA approved drug, an integrin antagonist used for
the treatment of cardiovascular disease (57). Whereas the
integrin-targeting sequence of each ligand is contained within
chemically similar, seven-membered, cyclic rings (57, 73), they
exhibited different activity profiles. For example, cHArGD and
eptifibatide, which share a common homoarginine residue, bound to
platelet
IIb
3 receptors with comparable
EC50 values, whereas cRGD bound an order of magnitude more
weakly. This pattern extended to the ability of each ligand to block
platelet aggregation, a fibrinogen-dependent function, as
well as to their effects on platelet:fibrin adhesion. Based on its
affinity and fibrin selectivity, cHArGD was used for most subsequent
IIb
3 ligand binding studies; a minimum
5-fold molar excess of ligand was used to achieve >95% receptor
saturation.
Biological properties of integrin ligands
Ligand Binding Effects on Global Stability of the
IIb
3
Tryptophan fluorescence emission spectra were recorded as a
function of temperature from 20 to 60 °C for both free
IIb
3 and the
IIb
3·cHArGD complex to discern the
effects of ligand binding on the thermal stability of the receptor (31,
50). During heating,
max shifted from 342 ± 2 nm at
20 °C to 337 ± 2 nm at 60 °C for the ligand-free integrin;
in the presence of cHArGD, a shift from 340 ± 3 to 338 ± 2 nm was observed (n = 3). These data were expressed as
the ratio of the background-subtracted emission at 350 nm to that at
320 nm, F350/F320, thus
correcting for the inherent temperature-dependent decrease
in protein fluorescence emission (60).
As illustrated in Fig. 1 (open
circles), in the absence of ligand, the
F350/F320 ratio for the
IIb
3 complex exhibited a sigmoidal
temperature dependence, decreasing by 25% from 20 to 60 °C with a
midpoint, Tm = 44 °C. Data obtained in three such
experiments yielded a 22 ± 1% decrease and Tm = 41 ± 3 °C. In contrast, an approximately linear 8 ± 3%
decrease in the F350/F320
ratio resulted for the ligand-bound receptor (solid
circles). These data indicate that ligand binding alters the
response of the integrin to increased temperature. However, the
temperature-induced changes in emission ratio determined with both free
and bound receptors were not readily reversible as indicated by data
obtained during cooling cycles (light gray triangles,
IIb
3; dark gray triangles,
IIb
3:cHArGD).
|
It is important to emphasize that our data ("Experimental
Procedures") and that of Aoudia and Zana (40) demonstrate that the
physical properties of octyl glucoside micelles are nearly invariant
from 20 to 60 °C, so that the effects we observed are not artifacts
related to changes in micelle structure, bur rather reflect
temperature- and ligand-induced changes in the physical state of the
IIb
3 integrin. However, because
blue-shifted fluorescence emission spectra are not expected for thermal
denaturation (50), we investigated the basis for this thermal
transition by additional biophysical techniques.
Ligand Binding Effects on the Secondary Structure of
IIb
3
Circular dichroic spectra were obtained for
IIb
3 to determine the extent of thermal
unfolding over the interval 20-60 °C. At 20 °C both free
receptor and the
IIb
3·cHArGD
complex exhibited similar minimum molar ellipticities of
approximately
6000 to
7000 degree/mol at ~208-211 nm, followed
by a sharply increasing positive signal at lower wavelengths. However,
because of the high absorbance of the octyl glucoside required for
receptor solubilization, reliable data were not obtained below 205 nm,
thus precluding deconvolution of the spectra to obtain estimates of
secondary structure (75). Therefore, an empirical approach was
followed, based on the observation that the difference signal between
native
IIb
3 and that unfolded in
guanidinium chloride was greatest at 222 nm (29). As shown in Fig.
2 (open circles), the
[
]222 for
IIb
3 exhibited
a sigmoidal decline with increasing temperature. The 47 °C midpoint
of this transition was similar to that observed by fluorescence.
Results from three experiments yielded Tm = 45 ± 2 °C and an 18 ± 1% signal change. A linear decrease in [
]222 of 9 ± 1% was observed for the
IIb
3·cHArGD complex, similar to the
results of the fluorescence experiments in Fig. 1. These thermally
induced molar ellipticity changes were not fully reversible, as the
data in Fig. 2 also indicate (gray triangle,
IIb
3; dark gray triangle,
IIb
3·cHArGD). As was the case for the
fluorescence data, the signal changes obtained by circular dichroism
are opposite in direction to those expected for unfolding (29). Thus
these data indicate that
IIb
3 undergoes
temperature-driven conformational changes that do not reflect global
unfolding. Furthermore, ligand binding changes the conformational
response of
IIb
3 to increased temperature.
|
Effects of Ligand Binding and Temperature on Integrin Oligomerization
Dynamic Light Scattering--
Recognizing the possibility that
changes in integrin oligomerization could have contributed to the
thermal transitions observed by fluorescence and circular dichroic
spectroscopy, a series of dynamic light scattering experiments (28) was
initiated to characterize the quaternary structure of the integrin at
two temperatures, 20 and 40 °C. As illustrated in Fig.
3, the free
IIb
3 integrin (white bars)
exhibited a stable, narrow size distribution centered at ~14 nm
diameter. Following a 4-h incubation at 40 °C, the peak shifted to
~32 nm (black bars). A small quantity of material, <1%
by weight, was also present at diameters >500 nm (data not shown).
Data obtained with buffered octyl glucoside yielded a peak diameter of
5 nm at 20 °C and 5-6 nm at 40 °C (data not shown), again
indicating that changes in OG micelle size were unlikely to make a
significant contribution to the these observations.
|
We next turned our attention to the effects of ligand binding on the
quaternary structure of IIb
3. One hour
after addition of excess ligand-mimetic peptide cHArGD to a sample of
IIb
3 integrin at 20 °C, the peak of
the size distribution shifted to ~30 nm (light gray bars).
An additional increase in peak diameter to ~47 nm was observed
following a 4-h incubation of the
IIb
3·cHArGD complex at 40 °C
(dark gray bars); ~1% of the material formed oligomers
>500 nm in diameter (data not shown). These results are representative
of the temperature- and ligand-induced changes observed by dynamic
light scattering in two experiments with
IIb
3 alone and three with
IIb
3·cHArGD complexes.
The light scattering data indicate that integrin oligomerization
increases at 40 °C, and that ligand binding further enhances self-association above that induced by increased temperature alone. Therefore, integrin oligomerization probably contributed to the temperature-induced, ligand-sensitive conformational changes in IIb
3 we observed by fluorescence and
circular dichroic spectroscopy. However, size distributions derived
from dynamic light scattering data are inherently sensitive to small
quantities of high molecular weight material because they return a
z-average profile (76). Therefore, we investigated this
issue with additional biophysical techniques.
Sedimentation Velocity--
Complementary information about the
effects of ligand binding and temperature on
IIb
3 self-association was obtained by
sedimentation velocity measurements that return a weight average size
distribution (77). Time derivative analyses (62) showed that at
20 °C the free integrin sedimented as a stable single species with
s20,w = 8.5 S, as indicated by the
open triangles in Fig. 4.
Evidence of substantial oligomerization was obtained after a 3-h
incubation at 40 °C (black triangles). A weight average
s20,w = 12.2 S was obtained, and the
distribution of sedimenting species extended out to 30 S. These data
are consistent with a mixture of protomers, dimers, trimers, and higher
order species (28). The
IIb
3·cHArGD
complex sedimented as a single 8.0 S species at 20 °C (light
gray circles); this shift toward a slower sedimenting species upon
ligand binding has been shown by us to reflect a ligand-induced conformational change to a more open
integrin conformation (28, 31). Following a 3-h incubation at 40 °C,
oligomers were also observed for the
IIb
3·cHArGD complex at 40 °C
(dark gray circles), although the distribution (weight
average s20,w = 9.7 S) was shifted
somewhat toward smaller oligomers compared with the ligand-free
integrin. Consideration of data obtained in replicate experiments
yielded a temperature-induced 43% increase in the weight average
s20,w for the free integrin but only a
22% increase for the ligand-bound form.
|
Whereas both sedimentation velocity and dynamic light scattering
demonstrated that increased temperature and ligand binding promoted
IIb
3 oligomerization, the size
distributions obtained by these techniques differed in their
quantitative details, as can be seen by comparing Figs. 3 and 4. For
example, DLS detected increased oligomerization for the
IIb
3·cHArGD complex at both 20 and
40 °C compared with the free receptor. Conversely, no
IIb
3·cHArGD oligomers were detected by
sedimentation velocity at 20 °C, and somewhat larger oligomers were
observed for the free receptor compared with the bound at 40 °C.
Because a substantial pressure gradient develops in a rapidly rotating
ultracentrifuge cell (79), we considered the possibility that
pressure-induced dissociation (79, 80) could have influenced the
distribution of integrin oligomers observed by sedimentation velocity.
Following the treatment presented by Kegeles et al. (79), we
estimated that the pressure reached 100 atmospheres near the cell
bottom during a sedimentation velocity experiment at 35,000 rpm.
Therefore, we addressed this issue by examining the distribution of
sedimenting species as a function of sedimentation time, based on the
reasoning that pressure-induced dissociation would become more
pronounced later in the run. However, examination of g(S) profiles for
the IIb
3·cHArGD complex at 20 °C
such as that presented in Fig. 4, revealed a set of single, symmetric profiles that exhibited the expected peak sharpening with time (62). We
did not observe any significant accumulation of sedimenting material in
the 5-6 S range expected for the dissociated
IIb and
3 subunits. Whereas the patterns obtained at 40 °C
were more complex because of the increased presence of oligomeric
species, evidence for pressure-induced dissociation was not observed
there either.
These findings are in keeping with the observation that the sedimentation coefficients calculated with the Svedberg equation (60) using weight average molecular weights obtained from our classical light scattering data and z-average translational diffusion coefficients obtained from our dynamic light scattering data are in approximate, although not quantitative, agreement with those obtained directly by sedimentation velocity (data not shown). Given the differential sensitivity of these two hydrodynamic techniques to macromolecular size distributions (81, 82), we investigated this issue in more detail by a thermodynamic approach, sedimentation equilibrium (64).
Sedimentation Equilibrium--
Changes in the molecular weight
distribution of IIb
3 were determined by
sedimentation equilibrium as a function of temperature and ligation
state. Data presented in Fig. 5 depict
the weight average molecular weight, Mw,
versus temperature for free
IIb
3 (open triangles), as well
as its complexes with three integrin ligands: cHArGD (solid
circles), cRGD (gray squares), and eptifibatide (dark gray diamonds). In the absence of ligands,
IIb
3 exhibited Mw
values ranging from 210,000 to 247,000 over the range
20-35 °C, consistent with the 232,000 expected for the integrin
protomer (dashed line). However, substantial molecular
weight increases, to ~400,000 and 370,000, were observed at 37 and
40 °C, respectively, indicative of temperature-induced aggregation
for the ligand-free integrin (64). In contrast,
Mw data obtained for the ligated integrins
showed a more gradual 1.6-fold increase over the range 20 to 40 °C
(solid line), indicating the data obtained for the ligated
integrin were more consistent with oligomerization, rather than the
aggregation behavior observed for the free receptor.
|
Evidence for Entropy-driven Oligomerization
Van't Hoff Analyses--
Sedimentation equilibrium data obtained
with the integrin-ligand complexes were analyzed further by nonlinear
regression (63) to extract a set of dimerization constants for each
complex as a function of temperature. The resultant parameters are
plotted in Fig. 6 as ln Ka
versus 1/T. Following the Van't Hoff equation,
|
![]() |
(Eq. 1) |
|
Electron Microscopy Studies of the Effects of Temperature and
Ligand Binding on Oligomerization of
IIb
3--
The preceding results are
supported by examination of
IIb
3 by
electron microscopy in the presence and absence of cHArGD and at 20 or
37 °C. Following a 3-h incubation in HSC-OG buffer, complexes of
IIb
3 were diluted into 0.05 M
ammonium formate at pH 7.4, 30 mM octyl glucoside, 15%
glycerol to a final concentration of 20-25 µg/ml. The samples were
examined by transmission electron microscopy following rotary shadowing
with tungsten. Glycerol is necessary to prevent surface artifacts, but
the concentration was lowered relative to that used in previous
experiments, because we found that the size distribution of
IIb
3-ligand complexes shifts toward
larger oligomers in the presence of 30% glycerol (28, 31).
Images obtained from samples prepared at 20 °C in 15% glycerol
showed almost entirely monomeric forms of
IIb
3, which are illustrated in previous
papers (28, 31). The monomers consist of a globular head region with
two long tails extending from one side. The tails often appear to be
connected to each other at their distal ends. In samples prepared at
37 °C, there was a striking change in the complexes, in that many of
them were clustered into oligomers. The oligomers have a specific
structure, such that the complexes appear to be interacting with each
other via their tails. Dimers are present as two complexes interacting
via their tails, with their heads at opposite ends, such that the two
complexes are related by a 180° rotation about an axis perpendicular
to the page at the center of the complex (Fig.
7A). Larger oligomers are seen
as clusters with their tails in the center and their heads at the
periphery, a structure with the appearance of a "rosette" (Fig.
7A). Oligomers with similar structures were observed with
IIb
3 samples incubated in the presence of
excess cHArGD peptide at 37 °C (Fig. 7B).
|
The effects of temperature and ligand binding were quantified by
counting the number of integrin molecules present as
IIb
3 monomers, dimers, and higher-order
oligomers. As shown in Fig. 8, 87% of
the ligand-free integrins were monomeric at 20 °C compared with only
56% at 37 °C (compare white and black bars).
In the presence of the cHArGD peptide, 68% of the integrins were
present as monomers at 20 °C (Fig. 8, light gray bars).
In contrast, with cHArGD peptide at 37 °C, only 41% were monomers
(Fig. 8, dark gray bars). Whereas these trends toward
increased oligomerization associated with both increased temperature
and ligand binding are striking, the results should be interpreted with
caution because of the difficulties of counting individual integrins
within these complex oligomers. Likewise, direct quantitative
comparison of these size distributions to those obtained by light
scattering and analytical ultracentrifugation are precluded by the
necessity to include glycerol in the sample preparations for electron
microscopy, a procedure we have shown to promote oligomerization (28,
31). Despite these reservations, the electron microscopy results
clearly reinforce the conclusion that both ligand binding and increased temperature promote
IIb
3
oligomerization.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This investigation provides new insights into two questions
related to the dynamic regulation of integrin function. How do elevated
temperatures, especially those in the physiological range, affect the
secondary, tertiary, and quaternary structures of
IIb
3? What intermolecular forces drive
integrin self-association?
Regarding the response of IIb
3 to
increased temperature, our spectroscopic probes of protein secondary
and tertiary structure have detected a transition at 43 ± 3 °C
for the ligand-free receptor, although not to an unfolded state. In
fact, this new thermostable conformer displays increased helicity, as
monitored by a deeper 222-nm trough in the circular dichroic spectrum
of
IIb
3, and a blue-shifted intrinsic
fluorescence emission spectrum. The direction of both changes is
opposite to that we and others (29, 31, 50) have described for
guanidinium denaturation of the
IIb
3 integrin. Our observations are also consistent with those of Makogoneko et al. (50) who reported that
IIb
3 was stable in octyl glucoside up to
at least 95 °C, based on fluorescence emission and scanning calorimetry measurements. Combining this information with the temperature-induced changes in receptor quaternary structure observed here by dynamic light scattering, sedimentation velocity, sedimentation equilibrium, and electron microscopy, we propose that the ligand-free
IIb
3 integrin undergoes conformational
changes and forms aggregates at temperatures above 35 °C.
Our integrated spectroscopic, hydrodynamic and thermodynamic approach also demonstrates that receptor occupancy by ligand mimetic peptides promotes oligomer formation at lower temperatures, and protects the bound receptor from the cooperative thermal transition seen with the free integrin. Complementary size distribution data obtained by electron microscopy reinforces the concept that ligand binding and increased temperature interact to promote integrin oligomer formation.
Concerning the intermolecular forces that drive integrin
oligomerization, the thermodynamic parameters determined in this investigation support the concept that entropically favorable hydrophobic interactions play a major role in
IIb
3 self-association. In particular,
van't Hoff analyses of the temperature-dependent dimerization constants, measured with
IIb
3 complexed with each of three ligand
mimetics, yielded a pattern of large positive enthalpy changes offset
by even larger positive entropy changes. As shown in Table II, we
obtained comparable
H and
S parameters with
two structurally similar cyclized ligands, cRGD and cHArGD, developed by Cierniewski et al. (73) as high affinity
analogs of the integrin-targeting regions found on the adhesive
proteins, fibronectin and fibrinogen. A similar pattern of positive
H and
S dimerization values also resulted
when the pharmaceutical integrin antagonist, eptifibatide, developed by
Scarborough et al. (30) as a fibrinogen mimetic, was bound
to
IIb
3. This pattern of entropy/enthalpy
compensation has been observed for other self-assembling, entropy-driven systems (37, 48, 78).
We note that the uncertainty associated with the thermodynamic
parameters cited in Table II may be because of several factors. First,
integrin self-association driven by increased temperature and ligand
binding has an irreversible component that becomes more pronounced at
temperatures above 35 °C, as considered under "Experimental
Procedures." Second, whereas we have treated the process as a
monomer-dimer equilibrium, oligomers larger than dimers are seen by
electron microscopy. We have previously found that the sampling
conditions required for microscopy, especially the presence of
glycerol, enhances IIb
3 aggregation (28,
31). Third, the effects of octyl glucoside micelles must be considered in interpreting our results. Our work and that of others (40) have
demonstrated that the physical properties of octyl glucoside micelles
are essentially constant across the temperature range explored, so it
is unlikely that detergent effects dominate our thermodynamic data.
This point is reinforced by the sensitivity of each of our
conformational reporters to the ligation state of
IIb
3, again an effect that cannot be
readily explained by changes in detergent properties.
Returning to a consideration of the structural basis for integrin
clustering, homology modeling (68), based on the recently published
ectodomain crystal structure of the closely related v
3 integrin (21), indicates that 19 of
the 24 tryptophan residues in the
IIb
3
complex are present in the ectodomain of the receptor. Hence,
conformational changes, such as ligation-linked perturbation of the
~1600 A2
/
subunit interface in the
v
3 integrin (21, 22), could shield some
of these ectodomain spectral reporters from solvent and contribute to
the observed blue-shifted emission. Whereas activation of integrin
2
1 involves ectodomain conformational changes that remove one helical turn and break a hydrophobic contact (74), neither of those effects would explain our results.
Conversely, there are three more tryptophan residues in the
transmembrane segment of
IIb and one each on
transmembrane and cytoplasmic regions of
3. Thus we
propose that thermally driven integrin oligomers are stabilized, in
part, by formation of helix bundles that bury key tryptophan residues
in transmembrane and cytoplasmic domains of
IIb
3. Electron microscopy reinforces this
concept by showing how oligomers of
IIb
3
are joined at their tails, an observation that may also explain their
increased helical CD signal and blue-shifted fluorescence spectra.
Evidence supporting a helix-bundling, integrin-clustering mechanism
also comes from recent biophysical characterizations of protein
fragments corresponding to integrin transmembrane and/or cytoplasmic
domains (6, 26, 72). The extent to which the integrin cytodomains alone
form ordered structures remains controversial, as Vinogradova et
al. (6) first reported CD and NMR evidence identifying a helical
segment in the membrane-proximal region of the IIb
subunit, when tethered to a lipid environment. Weljie et al.
(9) recently reported NMR data indicative of helical dimer interactions
for the membrane-proximal regions of the
IIb and
3 cytoplasmic domains. In contrast, Ulmer et
al. (72) found both cytoplasmic regions to be flexible and
unstructured in aqueous solution, although their NMR data did indicate
a helical structure could form within the tail of
3. Li
et al. (26) detected homomeric dimers and trimers with
constructs encompassing the
IIb and
3 membrane-spanning and cytoplasmic domains and proposed a role for these
interactions in integrin clustering.
Taken together, these observations support the postulate that integrin
self-association via its transmembrane and cytoplasmic domains could be
stabilized by new contacts between regions poised to interact in the
resting integrin protomers. This concept is illustrated schematically
in Fig. 9A with an image of an
integrin dimer assembled from a threading model of the
IIb
3 ectodomain, based on the crystal
structure of the
v
3 construct recently described by Xiong et al. (21). We have attached helical
transmembrane segments to the resultant ectodomain model; the
conformations of the cytodomain segments are based on NMR data and
secondary structure predictions (6, 72). We have chosen the extended version of the integrin model proposed by Xiong et al. (21), rather than the original bent structure that they observed for
v
3 in both the presence and absence of
ligand (21, 22). Our previous hydrodynamic and electron microscopy
data strongly indicate that the extended conformation is favored under
our experimental conditions (28, 31).
|
Whereas this schematic is designed to resemble the dimers
seen by electron microscopy, we propose that additional ectodomain interactions may be favored with integrins that reside on a cell surface. Tail to tail oligomers may form after an initial side by side
integrin self-association, followed by micelle fusion and rearrangement
to a more stable conformation induced by the high curvature of the
octyl glucoside micelle (31). We hypothesize that in vivo
both ligand binding and increased temperature favor the assembly of
integrin dimers that are stabilized by helix bundles formed from their
transmembrane (and possibly cytoplasmic) domains as well as through
additional contacts between their ectodomains (illustrated in Fig.
9B). These integrin clusters could enhance the local
concentration of integrin-associated cytoplasmic proteins, thus
insuring the rapid, efficient transmission of a signal reporting ligand
binding at their distant ectodomains to the interior of the cell.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. C. Stahle for expert technical assistance, Dr. J. B. Edelson for skilled editing, and Aaron E. Hantgan for graphic design skills.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant MCB-9728122 and the Venture Fund of Wake Forest University School of Medicine (to R. R. H.), National Institutes of Health Grant AI15892 (to D. S. L.), European Community Grant BI04-CT96-0662 (to M. R.), and National Institutes of Health Grant HL 30954 (to J. W. W.).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.: 336-716-4675; Fax: 336-716-7671; E-mail: rhantgan@wfubmc.edu.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M208869200
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ABBREVIATIONS |
---|
The abbreviations used are:
OG, n-octyl--D-glucopyranoside;
cHArGD, cyclo(S,S)-L-lysyl-L-tyrosyl-glycyl-L-cystinyl-L-homoarginyl-glycyl-L-aspartyl-L-tryptophanyl-L-prolyl-L-cystine;
cRGD, cyclo(S,S)-L-lysyl-L-tyrosyl-glycyl-L-cystinyl-L-arginyl-glycyl-L-aspartyl-L-tryptophanyl-L-prolyl-L-cystine;
Mw, weight average molecular weight.
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