The Disintegrin Echistatin Stabilizes Integrin αIIbβ3's Open Conformation and Promotes Its...

doi:10.1016/j.jmb.2004.08.009 J. Mol. Biol. (2004) 342, 1625–1636

The Disintegrin Echistatin Stabilizes Integrin aIIbb3’sOpen Conformation and Promotes Its Oligomerization

Roy R. Hantgan1*, Mary C. Stahle1, John H. Connor1, Douglas S. Lyles1

David A. Horita1, Mattia Rocco2, Chandrasekaran Nagaswami3

John W. Weisel3 and Mary Ann McLane4

1Department of BiochemistryWake Forest University Schoolof Medicine, Winston-SalemNC 27517, USA

2Medicina Rigenerativa, IstitutoNazionale per la Ricerca sulCancro, Genova I-16132, Italy

3Deparment of Cell andDevelopmental BiologyUniversity of PennsylvaniaPhiladelphia, PA 19104, USA

4Department of MedicalTechnology, University ofDelaware, Newark, DE 19716USA

0022-2836/$ - see front matter q 2004 E

Abbreviations used: GST, glutathPRP, platelet-rich plasma; GFP, gel-E-mail address of the correspond

[email protected]

We have employed echistatin, a 5.4 kDa snake venom disintegrin, as amodel protein to investigate the paradox that small ligand-mimetics canbind to the resting aIIbb3 integrin while adhesive macromolecules cannot.We characterized the interactions between purified human aIIbb3 and tworecombinant echistatin variants: rEch (1-49) M28L, chosen for its selectivitytoward b3-integrins, and rEch (1-40) M28L, a carboxy-terminal truncationmutant. While both contain an RGD integrin targeting sequence, only rEch(1-49) M28L was an effective inhibitor of aIIbb3 function. Electronmicroscopy of rotary shadowed specimens yielded a variety of aIIbb3conformers ranging from compact, spherical particles (maximum dimen-sion 22 nm) to the classical “head with two tails” forms (32 nm). Thepopulation of larger particles (42–56 nm) increased from 17% to 28% in thepresence of rEch (1-49) M28L, indicative of ligand-induced oligomeriza-tion. Sedimentation velocity measurements demonstrated that both fulllength and truncated echistatin perturbed aIIbb3’s solution structure,yielding slower-sedimenting open conformers. Dynamic light scatteringshowed that rEch (1-49) M28L protected aIIbb3 from thermal aggregation,raising its transition mid-point from 46 8C to 69 8C; a smaller shift resultedwith rEch (1-40) M28L. Sedimentation equilibrium demonstrated that bothechistatin ligands induced substantial aIIbb3 dimerization. van’t Hoffanalysis revealed a pattern of entropy/enthalpy compensation similar totirofiban, a small RGD ligand-mimetic that binds tightly to aIIbb3, butyields smaller conformational perturbations than echistatin. We proposethat echistatin may serve as a paradigm for understanding multidomainadhesive macromolecules because its ability to modulate aIIbb3’s structureresides on an RGD loop, while full disintegrin activity requires an auxiliarysite that includes the carboxy-terminal nine amino acid residues.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: integrin; disintegrin; conformation; oligomerization;thermodynamics
*Corresponding author
Introduction

Integrin regulation of cellular adhesiveness iscentral to physiological processes ranging fromembryonic development to programmed cell death,and aberrant integrin activation contributes tothrombosis, cancer and inflammatory disorders.1

However, the molecular mechanisms that convert

lsevier Ltd. All rights reserve

ione-S-transferase;filtered platelets.ing author:

resting integrins into dynamic receptors, capable ofsensing and responding to changes in a cell’sexternal environment, remain a matter of intenseinvestigation and some controversy.2–4 The contro-versy arises in part from the divergent views of thestructural basis for integrin activation provided byX-ray diffraction crystallography,5,6 electronmicroscopy,7–10 and biophysical solution confor-mation probes.10–13 Resolution of this dilemma hasproven problematic because most studies describ-ing integrin–ligand interactions have used consti-tutively active ectodomain constructs.1,14,15 In aprevious study, Weisel et al.16 analyzed nativeaIIbb3 integrins bound to fibrinogen; however,

d.

1626 Echistatin: Integrin Conformation and Oligomerization

their study was not designed to elucidate themechanisms for ligand-linked conformationalchanges. Thus, we have chosen to revisit this issueusing echistatin as a model protein ligand.

The conventional view of integrin structure,based on electron microscopy images, showedintegrins as elongated molecules with an ovalhead from which two tails extend.7,17,18 This waschallenged when the first high resolution structureof an integrin ectodomain was published in 2001.5

Examination of the crystal structure of the extra-cellular domain of avb3 revealed a sharply bent,multidomain molecule whose V-shape was pre-served even when an Arg-Gly-Asp ligand wasdiffused into the crystals.5,6,19 While some haveargued that crystal packing artifacts limited therange of ligand-linked structural rearrangements,20

Xiong et al.2 have proposed a “deadbolt” modelwhere reversible inter-domain contacts within thebent conformer regulate avb3 binding function.Small angle X-ray scattering data recently reportedby Mould et al.21 on an a5b1 ectodomain constructdemonstrated that no large integrin conformationalchanges took place upon binding a fibronectinfragment (Fn 6-10). The combination of X-raydiffraction and X-ray scattering data would seemto indicate that integrin activation or “priming”3

does not require large conformational changes, butthis concept is not universally accepted.

In contrast to the minimalist view, Takagi &Springer have proposed a “switchblade” mecha-nism of integrin activation22 based on electronmicroscopy and biophysical data, as well asmutationally engineered structural constraints andperturbations.8,23–25 Takagi et al.26 also presentedelectron microscopy images of an a5b1 ectodomain:fibronectin fragment (Fn 7-10) complex with apronounced separation in the integrin’s remnantstalks. Evidence that these ectodomain confor-mational changes may propagate across the plasmamembrane to an integrin’s cytoplasmic regionscomes from in vivo FRET measurements that showa O100 A cytodomain separation on aLb2 integrin-cyan (or yellow) fluorescent protein constructs,activated by either inside–out or outside–insignaling.27

Both the deadbolt and switchblade modelsprovide an attractive mechanism to explain integrinactivation, but a note of caution is in order becausethe barriers that prevent resting, full-length integ-rins from binding macromolecular ligands have yetto be fully elucidated. For example, Adair & Yeager9

used electron cryo-microscopy to obtain a 20 Aresolution structure for the intact aIIbb3 complex,isolated and imaged in octyl glucoside micelles. Theresulting model of the ligand-free integrin exhibiteda large ectodomain connected by a narrow rod-likeputative transmembrane domain to a small bilobedsegment, assigned to the aIIbb3 cytodomains. Theirmodel was more compact than the extendedintegrins typically observed by TEM,7,10,12 yet itsectodomain appeared less severely bent than theavb3 crystal structure.5 Concerning integrin–ligand

interactions, Adair & Yeager also reported electro-phoretic mobility data indicating that echistatin, a5.4 kDa RGD-containing disintegrin polypeptidefound in Echis carinatus venom,28–31 induced atransition in aIIbb3 from a low to a high-affinitystate.9 However, they did not present any images ofthe integrin–echistatin complex.

We have also employed an integrated biophysicaland electron microscopy approach to describe thelinks between ligand binding and integrin confor-mational changes.10,12,13,32 Hence, we recognized inechistatin the opportunity to critically evaluate themechanisms for integrin activation using both afull-length integrin and a protein ligand, rather thanligand-mimetics whose binding modes may notfully reflect the intricacies of aIIbb3’s interactionswith its physiological binding partners. Echistatin isa model protein that presents both a paradox anda paradigm for understanding the regulation ofintegrin function. The paradox comes from theobservation that echistatin binds with comparableaffinity to aIIbb3 complexes on both resting andstimulated human blood platelets,33 whereasadhesive proteins such as fibronectin and fibrino-gen display high-affinity interactions only with the“activated” receptor.34 The paradigm derives fromthe common RGD–integrin targeting sequenceshared by echistatin and aIIbb3’s primary physio-logical ligands.35,36 In the experiments presentedhere, we will demonstrate that echistatin perturbsthe resting receptor, and stabilizes an open con-former with an enhanced, entropy-driven oligo-merization constant. We will show that echistatin’sability to modulate aIIbb3’s solution structureresides primarily within the RGD segment thatprotrudes from its disulfide-bonded core, while fulldisintegrin activity requires its carboxy-terminalsegment.

Results

Recombinant echistatin variants and theirbiological activities

Echistatin is a member of the disintegrin family oflow-molecular mass proteins that employ an Arg-Gly-Asp integrin targeting sequence to bind to andblock integrin function.37 A nearby methionineresidue also contributes to echistatin’s selectivityfor certain integrins, as mutation to leucine dis-rupted binding to a5b1, but had no effect on bindingto the b3 integrins.38 In addition, echistatin has aputative auxiliary binding site at its carboxyterminus.39,40 Thus, we examined the interactionsof full-length (rEch (1-49) M28L) and truncated(rEch (1-40) M28L) recombinant echistatin vari-ants41 with the purified aIIbb3 integrin, isolated in afunctional conformation in octyl glucosidemicelles.10,11,13

We assessed the ability of each recombinantechistatin variant to inhibit platelet aggregationand platelet adhesion to fibrin, two activities that

Echistatin: Integrin Conformation and Oligomerization 1627

are dependent on the aIIbb3 integrin.32,42,43 rEch(1-49) M28L exhibited an IC50Z126(G37) nM forinhibition of ADP-stimulated platelet aggregation, aparameter that agreed well with the values of136(G29) nM and 124(G18) nM reported byWierzbicka-Patynowski et al.38 for native andrecombinant echistatin (1-49), respectively. In con-trast, rEch (1-40) M28L had no effect on plateletaggregation at concentrations ranging from 100 nMto 1000 nM. rEch (1-49) M28L also proved to be aneffective inhibitor of platelet adhesion to clottedfibrin,32 yielding IC50Z124(G88) nM. Considera-tion of the platelet count and integrin receptordensity32 indicates that rEch (1-49) M28L caused a50% reduction in platelet adhesive function at afour- to sixfold molar excess over its target, theaIIbb3 integrin. In fact, the effectiveness of rEch(1-49) M28L in blocking platelet–fibrin interactionswas comparable to the cyclic peptide integrinantagonists cHArGD and eptifibatide,13 andexceeded that of the ligand-mimetic tirofiban.32

Effects of recombinant echistatin on aIIbb3structure examined by transmission electronmicroscopy

The effects of full-length echistatin on aIIbb3conformation and oligomerization were probed byexamining rotary shadowed samples of the free andligand-bound receptors by transmission electronmicroscopy. We observed a variety of aIIbb3conformers, in both the absence and presence ofrEch (1-49) M28L, ranging from compact, sphericalparticles to the classical headwith two tails forms.7,10

Some of these particles appeared to have separatenodules corresponding to either a split in the headdomain, as we reported earlier in the presence ofligand-mimetic peptides,10,12 or separate stalks, asothers have described.8,27,44 Integrin dimers, joinedthrough tail-to-tail contacts similar to thoseobserved with aIIbb3 and synthetic ligands,10–13,32

or Mn2C,44 were also present. Examples of the mostcommon forms observed are illustrated in Figure 1;however, this image is not meant to represent thefrequency of occurrence of these particles.

Given this multiplicity of integrin morphologies,we measured the distribution of particle sizes forboth free and bound receptors, using the maximumlinear dimension of each object as an index(SigmaScan; Jandel Scientific). As indicated in thehistograph in Figure 1, comparable size distri-butions resulted for both free and bound integrins.The free aIIbb3 complex exhibited a mean particlelength of 30.7(G9.7) nm (NZ677); 31.8(G9.7) nm(NZ622) was obtained in the presence of rEch (1-49)M28L. While the difference between these meanparticle sizes was small, it did achieve statisticalsignificance (pZ0.02, t-test45).

Further interpretation of these data was facili-tated by comparing these parameters to models ofthe full-length aIIbb3 complex in conformationsranging from bent,5,9 to fully extended.8,13,27 Thesurface representations of each model presented in

Figure 1 were prepared, and their maximum lineardimensions calculated, using the moleculargraphics package MolMol.46 The experimentallydetermined mean particle dimensions (31–32 nm)correspond quite closely to the calculated para-meters of 31 nm and 32 nm for the extended andstalk-split models, respectively. Modeling demon-strated that docking echistatin to aIIbb3’s ecto-domain increased these calculated maximumparticle lengths by w1 nm, an observation thatmay also explain the experimentally determineddistribution means for aIIbb3 alone and in thepresence of echistatin.To explore this issue further, we fitted the

experimental data obtained with aIIbb3 alone to asum of Gaussian distributions using an automatedpeak finding algorithm (PeakFit; Systat Software,Inc.). This procedure identified a major peak at32 nm and smaller peaks at 20, 44 and 56 nm, asillustrated by the black lines in the histographshown in Figure 1. (A minor peak at w3 nm, mostlikely octyl glucoside micelles,10 was omitted fromthe fitting procedure.) A similar pattern of peaks:32, 22, 42, and 54 nmwas detected in the presence ofechistatin (gray lines). Systematically removingthese peaks resulted in a two- to fourfold increasein the fit standard error, a goodness-of-fit parameterthat accounts for changes in the degrees of free-dom.45 Therefore, we concluded that the four-peakfit best described the experimental data. Interest-ingly, the population tentatively identified as bentconformers (20–22 nm) was essentially unchangedin the presence (12%) or absence (13%) of echistatin,while the population of extended integrins (32 nm)decreased from 81% to 68% and the oligomerpopulation (42–56 nm) increased from 17% to 28%.Interpretation of these data was aided by themodels of tail-to-tail integrin oligomers (calculatedlength 50 nm) shown in Figure 1.These observations suggest that echistatin pro-

motes integrin oligomerization, so to clarify thisissue, we also examined echistatin’s effects on theaIIbb3 solution conformation with three comple-mentary biophysical techniques that we found to besensitive indicators of tertiary and quaternarystructural changes induced by synthetic integrinligands: sedimentation velocity, dynamic light-scattering, and sedimentation equilibrium.10–13,32

Effects of recombinant echistatin variants onaIIbb3 solution structure

We have previously demonstrated that ligand-mimetics that share with echistatin an RGDintegrin-targeting sequence perturb the confor-mation of the purified aIIbb3 integrin, yieldingan “open” conformer with increased frictionaldrag.10,32 Hence, we employed our most sensitiveconformational probe, sedimentation velocitymeasurements coupled with time-derivative ana-lyses,47 to determine the effects of rEch (1-49) M28Land rEch (1-40) M28L on aIIbb3 solution structure.As demonstrated in Figure 2(A), an equimolar

Figure 1. Transmission electron microscopy of rotaryshadowed integrin in the presence and absence ofechistatin; micrographs, models and measurements.Image gallery: (A) control integrin aIIbb3, which consistsof a globular head-piece and two tails, usually touchingeach other. In all experiments a variety of structures wereobserved, but only examples of the most commonlyobserved forms are shown here. (B) Integrin aIIbb3 in thepresence of echistatin. Again, a variety of structures wereobserved, but some examples of the most commonlyobserved forms are shown. Integrin complexes have thesame general appearance as the controls but the totallength of most molecules is slightly longer. (C) Examplesof dimers of aIIbb3 in the presence of echistatin. It isimportant to note that dimers are present in the controlintegrin and integrin with echistatin, but only a smallfraction of integrins are present as dimers. Integrinmodels: the panel below shows models (and maximumdimensions) of the full-length aIIbb3 complex, includingbound octyl glucoside micelles, corresponding to the


concentration of rEch (1-49) M28L shifted aIIbb3’sg(S) profile (open circles) toward a slower-sedi-menting conformer (gray circles); comparableeffects were observed at a tenfold molar excess(filled circles). The effects of the truncated echistatinwere somewhat less pronounced when present atan equimolar concentration, but were similar to thefull-length disintegrin at a tenfold molar excess(Figure 2(B)).

Preliminary hydrodynamic computations usingthe SOMO algorithms48–50 indicate that a restingintegrin–echistatin complex would exhibit a sedi-mentation coefficient w2% larger than the ligand-free integrin. In contrast, computations based onmodels like those presented in Figure 1 indicate thatligand-induced structural asymmetry woulddecrease the receptor’s sedimentation coefficientby some 4–12%. As illustrated by the symbols atthe top of Figure 2(A), SbentOSpartially extendedOSfully extended/echistatinOSstalk split/echistatin. Thus, theexperimentally determined decreases in S are mostlikely due to conformational changes in the receptorlinked to echistatin binding.

Examination of a range of echistatin concen-trations demonstrated a 7.2(G1.3)% decrease inaIIbb3’s sedimentation coefficient for rEch (1–49)M28L and a comparable 5.5(G1.8)% decrease forrEch (1–40) M28L (Figure 2(C)). These data indicatethat echistatin’s 11 residue carboxy-terminal seg-ment is not required for the ligand to perturbaIIbb3’s solution conformation. Consideration ofthe complete data set demonstrated that the effectsof both rEch (1-49) M28L and rEch (1-40) M28L onaIIbb3’s sedimentation profile were statisticallysignificant (p!3!10K3 in both cases; t-test45). Theeffects of rEch (1-49) M28L on aIIbb3 conformationwere not readily reversible, as removing excessligand following a one hour incubation still yieldeda slower-sedimenting conformer (Figure 2(C), opencircle). These observations provide direct evidencethat binding a single-domain protein ligand to afull-length integrin yields a species with a confor-mation distinct from that of the free receptor.

following structures: the avb3 ectodomain crystallo-graphic structure (open circle),5 the aIIbb3 cryo-electronmicroscopy 3D model (filled triangle),9 our extendedaIIbb3 model (open square),13 and the cytoplasmicdomain separation model (filled diamond) proposed bySpringer and colleagues.8,27 Our model of a tail-to-tailintegrin dimer13 is denoted by a filled circle. Thecalculated maximum dimensions of each of these modelsare indicated by the symbols at the top of the Figurebelow. Integrin dimensions: The histograph depicts thedistribution of integrin particle sizes observed in theabsence (open bars) or presence (filled bars) of recombi-nant echistatin. The continuous lines define the peaksdetected by fitting the experimentally observed distri-butions to a sum of Gaussian peaks (Peak Fit, SystatSoftware, Inc.); the gray lines identify peaks in the controlintegrin samples and the black lines in the integrin–echistatin samples.

Figure 2. Effects of recombinant echistatin on aIIbb3solution conformation determined by sedimentationvelocity. (A) Data obtained for aIIbb3 alone (open circles),CrEch (1-49) M28L at an equimolar concentration of(gray circles), or a tenfold molar excess (filled circles)were analyzed with time-derivative software (DCDTC,J. Philo77) to obtain sedimentation coefficient distributionfunctions, g(s*) versus s*.47 Continuous lines were obtainedby fitting the resultant distribution functions to a singleideal species. Top symbols denote the computed sedi-mentation coefficients for the models illustrated in Figure1.49,50 (B) Sedimentation velocity data obtained withaIIbb3 alone (open triangles), CrEch (1-40) M28L at a1.5-fold (gray triangles), or a tenfold molar excess (blacktriangles). (C) Fractional change in sedimentation coef-ficient versus the concentration of Echistatin variant: rEch(1-49) M28L (filled circles) or rEch (1-40) M28L (graytriangles); the continuous line was determined by fittingto a hyperbolic inhibition model.10 The open circledenotes the effects of transient exposure to rEch (1-49)M28L.


Effects of recombinant echistatin variants onaIIbb3 thermal stability

The observation that rEch (1-49) M28L and rEch(1-40) M28L both perturbed aIIbb3’s solutionconformation led us to test the hypothesis that theintegrin–echistatin complexes would be resistant tothermal aggregation, an effect we have recentlydescribed for ligand mimetic peptides.13 Here weemployed dynamic light-scattering measurementsas the most direct approach toward determiningtemperature-induced changes in integrin quater-nary structure.Figure 3(A) depicts the results of particle size

measurements performed as a function of tempera-ture with aIIbb3 alone (open circles) and in thepresence of a threefold molar excess rEch (1-49)M28L (filled circles). The ligand-free integrin

Figure 3. Effects of recombinant echistatin on aIIbb3thermal stability determined by dynamic light-scattering.(A) Z-average macromolecular diameter measurementsperformed as a function of temperature are plotted foraIIbb3 alone (open circles), and CrEch (1-49) M28L at a3.3-fold molar excess (filled circles). The inset shows thederivative of the Z-average diameter with respect totemperature plotted versus temperature, dZ/dT. Thisprocedure enabled the location of a transition mid-pointat 46 8C for the free integrin (open circles); the integrin–echistatin complex exhibited a minor peak at 48 8C and amajor transition at 69 8C (filled circles). (B) Similarprocedures applied in separate experiments to aIIbb3alone (open triangles), CrEch (1-40) M28L at a tenfoldmolar excess (gray triangles) yielded transition mid-points of 46 8C and 59 8C, respectively (inset).

Figure 4. Effects of temperature and recombinant


exhibited a sigmoidal increase in particle diameter,increasing from w19 nm at 20 8C to a plateau atw35 nm at temperatures above 70 8C. In contrast,the integrin–echistatin complex exhibited a biphasicprofile. The transition mid-points were determinedby plotting dZ/dT, the incremental change inz-average particle size with temperature, as afunction of temperature, as shown in Figure 3(A),inset. This analysis identified a single TmZ46 8C foraIIbb3 alone. The aIIbb3: rEch (1–49) M28L sampleexhibited two peaks, corresponding to TmZ49 8Cand a more pronounced transition at 69 8C. Asimilar transition at 69 8C was observed in thepresence of an 11-fold molar excess of rEch (1–49)M28L (data not shown). In separate experiments,Tm values of 46 8C and 59 8C were obtained foraIIbb3 alone, and in the presence of a fivefold molarexcess of rEch (1-40) M28L (Figure 3(B)), respect-ively. When present at a 1.4-fold molar excess, rEch(1–40) M28L caused a smaller effect, yielding TmZ49 8C (data not shown). These data indicate thatwhile echistatin’s 11 residue carboxy-terminal seg-ment is not required for the ligand to bind to aIIbb3,it does contribute to the stabilization of its solutionconformation. These observations also provide thefirst evidence that binding a protein ligand to theaIIbb3 integrin yields a more thermostable con-former, one similar to that induced by lowmolecular mass ligands.13

echistatin on aIIbb3 oligomerization determined bysedimentation equilibrium. (A) Data obtained as afunction of temperature for aIIbb3 alone (open squares),CrEch (1-49) M28L (filled circles), CrEch (1-40) M28L(dark gray triangles) were analyzed with WinNONLIN53

to obtain weight-average molecular weight values (andtheir standard deviations) as a function of temperature.(B) Data for the integrin–echistatin complexes in (A) werealso analyzed with WinNONLIN to extract dimerizationconstants; the resultant Ka values (and their standarddeviations) are presented in a (linear) van’t Hoff plot ofln Ka versus 1/T from which thermodynamic parameterswere determined.13

Effects of recombinant echistatin variants onaIIbb3 oligomerization

Since oligomerization or clustering is consideredto play a major role in the regulation of integrinfunction,51 we tested the hypothesis that integrin:echistatin complexes would exhibit larger self-association constants than the ligand-free receptor.Following procedures optimized with syntheticpeptide ligands,13 we carried out sedimentationequilibriummeasurements at temperatures rangingfrom 20–40 8C, with aIIbb3 alone, and in thepresence of either rEch (1-49) M28L or rEch (1–40)M28L. As illustrated in Figure 4(A), the free integrin(open squares) was stable from 20–35 8C, but it thenexhibited a sharp, wtwofold increase in weight-average molecular weight (Mw) at 40 8C. In contrast,the echistatin:integrin complexes (filled circles, rEch(1-49) M28L; filled triangles, rEch (1–40) M28L) eachshowed substantial, but stable, oligomerizationacross the temperature range from 20–40 8C. Theresultant Mw value (3.35(G0.16)!105) obtained inthe presence of rEch (1-49) M28L across this intervalwas 53% higher than the calculated value for anaIIbb3 protomer (2.18!105); that obtained withrEch (1–40) M28L was 46% greater. These resultsindicate substantial ligand-induced oligomeriza-tion, although even at the highest temperatureexamined, the observed molecular masses fellbetween that of an aIIbb3 protomer and dimer. Wehave used sedimentation equilibrium measure-ments to quantify the effects of peptides andpharmaceutical integrin antagonists on aIIbb3’s

quaternary structure; however, echistatin’s effectsonMw exceed those of any synthetic ligand we haveexamined.10–13,52 Since these size distribution datawere obtained after the 20 hour period required toachieve sedimentation equilibrium,10 they report arelatively slow oligomerization process that followsthe ligand-induced initial conformational changesdetected by sedimentation velocity.

These data were also analyzedwith NONLIN53 toobtain values of Ka, the receptor dimerizationconstant, as a function of temperature13 for eachintegrin–echistatin complex. Addition of higher-order terms to the oligomerization model did notcause a significant improvement in the quality ofthe fit; therefore, we concluded that a monomer–dimer equilibrium was the simplest model consist-ent with the data. Consideration of this equilibrium,using our experimentally determined dimerizationconstants and the range of integrin concentrations


examined, indicates that at 20 8C, 35–45% (byweight) of the integrins will be present as dimers,while at 40 8C, this value will rise to 50–60%. Figure4(B) depicts the results of van’t Hoff analysis, i.e. aplot of ln Ka versus 1/T, from which the followingthermodynamic parameters for oligomerization ofthe integrin: echistatin complexes were determined:DHZ9.3(G4.5) kcal/mol and DSZ55(G15) cal/deg-mol for rEch (1-49 M28L) and DHZ11.6(G5.0) kcal/mol and DSZ62(G16) cal/deg-mol for rEch (1-40M28L). These data indicate that echistatin’s 11residue carboxy-terminal segment is not requiredfor the ligand to promote aIIbb3 oligomerization.While the small slope of the van’t Hoff plots givesrise to a large uncertainty in the enthalpy data, thispattern of favorable oligomerization entropy partlyoffset by an unfavorable enthalpy change obtainedwith the aIIbb3–echistatin complexes is comparableto the effects of synthetic integrin ligands.13 Ourdemonstration that echistatin binding promotesaIIbb3 oligomerization expands our understandingof the mechanisms that regulate integrin functionby demonstrating that a single-domain proteinligand can modulate an integrin’s quaternarystructure, in a manner similar to that of lowmolecular mass integrin antagonists.13,32

Discussion

This investigation provides insights into thedynamic regulation of integrin function by demon-strating that echistatin binding to the aIIbb3integrin stabilizes an open conformer with anenhanced, entropy-driven oligomerization con-stant. We propose that despite its small size,echistatin can serve as a paradigm for under-standing much larger adhesive macromoleculesbecause its ability to modulate aIIbb3’s structureresides on an RGD loop, while full disintegrinactivity requires an auxiliary site. UnderstandingaIIbb3 integrin structure and function has provenparadoxical because low molecular mass ligand-mimetics (!1500 Da) can readily bind to the restingreceptor, yet physiological ligands, such as the340,000 Da adhesive protein fibrinogen, cannot. Bydemonstrating that recombinant echistatin can bindto aIIbb3, isolated without prior exposure to anyactivating ligands,11 we have extended the recep-tors’ “size exclusion limit” to at least 6 kDa.

We next turned to the question: what confor-mational changes are linked to echistatin binding tothe resting aIIbb3 integrin? Hydrodynamic compu-tations48–50 predict that echistatin binding alonewould cause an w2% increase in aIIbb3’s sedimen-tation coefficient, whereas ligand-induced asym-metry would decrease the S value from 4–12%.Because our experimental data demonstrate anw7% decrease in S value for the aIIbb3:rEch(1–49) M28L complex (Figure 2), we conclude thatechistatin binding is linked to a substantial confor-mational change in aIIbb3’s tertiary structure. Ourobservation that rEch (1-40) M28L yielded an w5%

decrease in S value indicates that the 11 residuespresent on echistatin’s flexible carboxy terminus30

are not required for this interaction. It is also likelythat the conformational plasticity inherent inintegrin structure,4,5,54–56 a concept reinforced byour electron microscopy observations of a range ofintegrin sizes and shapes (Figure 1), contributed tothe effects of echistatin binding on aIIbb3’s sedi-mentation profiles.While we have previously proposed an integrin

activation scheme in which ligand mimetic bindingis linked to subunit separation in aIIbb3’s headregion,10,12 we now recognize that such a model isincompatible with high resolution structural datathat demonstrate short-range electrostatic inter-actions occur between a cyclic RGD ligand andboth subunits in the avb3 complex.6 Hence, wehave sought alternative explanations for our obser-vations. For example, If echistatin binding toaIIbb3’s ectodomain were to induce a confor-mational change that propagates “downward”leading to either release the “deadbolt” proposedto lock an integrin into its bent conformation,2 or todisrupt interactions between its cytoplasmicdomains,27 the resultant increase in hydrodynamicvolume could account for the slower-sedimentingconformers.57 The magnitude of these echistatin-induced conformational perturbations are compar-able to those we have obtained with linear RGDXpeptides,10 but are two- to threefold greater than theeffects of tirofiban,32 a non-peptide integrinantagonist designed to replicate the RGD spatialcharge. Tirofiban’s smaller size and higher affinityfor the aIIbb3 integrin32 could explain why itseffects on aIIbb3 conformation were smaller thanechistatin’s. We may speculate that echistatinoperates by an induced fit mechanism,58 perturbingthe receptor’s conformation to maximize electro-static contacts with its target integrin15,39,40,59 at theexpense of binding energy.We have also demonstrated that echistatin bind-

ing is linked to increased integrin oligomerization,as evidenced by sedimentation equilibriummeasurements (Figure 4). We may speculate thatthe conformational perturbation initiated by echis-tatin binding at the aIIb/b3 subunit interfacepropagates from the receptor’s ectodomain throughits stalks, resulting in a disruption of contactsbetween its distant transmembrane and cyto-plasmic regions. In that case, the now unpairedtransmembrane segments from two separate integ-rins could associate to form oligomers. Ligand-induced stalk separation has also been described forthe ectodomain of the avb3 integrin,8 and breakingaIIb/b3 transmembrane interactions has beenimplicated in receptor oligomerization.44 Further-more, signaling mechanisms involving activation-induced disruption of cytodomain contacts on theaLb2 have recently been reported on living cells.27

However, one may question whether thermo-dynamic or kinetic barriers to micelle fusion couldhamper transmembrane association in a detergentenvironment.60 Resolving this issue experimentally

Figure 5. Summary of thermodynamic parameters forligand-linked integrin oligomerization. The height of eachbar denotes the magnitude of the free energy change,KDG (blue/purple color ramp), the enthalpy change, DH(green/yellow color ramp), or the temperature-weightedentropy term, TDS (brown/yellow color ramp) for thetwo echistatin variants examined here, as well as data forthe ligand-mimetic tirofiban and thepeptide ligand,eptifibatide, determined in our earlier work.13,32


is not straightforward, as the free energy changesassociated with transmembrane helix dimerizationin detergent micelles are quite sensitive to thedetergent concentration.61 In fact, Gottschalk et al.60,62

have proposed an alternate integrin activationscheme in which contacts between the aIIb and b3transmembrane segments are preserved duringactivation, while a scissors-like movement disruptsinteractions between the cytoplasmic domains thatstabilize the resting integrin conformation. On theother hand, Weisel and colleagues first observedtransmembrane separation with aIIbb3–fibrinogencomplexes,16 and we subsequently reported theformation of tail-to-tail integrin oligomers in thepresence of synthetic ligands.10,12,13 Recently, Li etal.44 described a similar aIIbb3 tail separation andoligomerization process induced by divalentmanganese cations. While all experiments reportedhere were carried out in 2 mM CaCl2, we haveobtained preliminary data indicating that aIIbb3exhibits a similar distribution of sedimentingspecies and weight-average molecular mass in2 mM MnCl2. All these studies were performedwith integrin receptors solubilized in octyl gluco-side micelles. Moreover, proteins have been crystal-lized in octyl glucoside micelles yielding highresolution structural data for oligomers composedof 2,63 6,64 and 1165 transmembrane helices. Theoctyl glucoside molecules typically bound perpen-dicular to the helix axis, making direct contact withnon-polar residues.63,65 We have incorporated thesedetergent-binding characteristics into the modelswe have developed to predict the hydrodynamicparameters of full-length aIIbb3 integrin protomersand dimers.13

The entropy-driven nature of echistatin-induceddimerization supports the concept that interactionsbetween the receptors’ hydrophobic transmem-brane segments contribute to oligomerization. Infact, the magnitude of the favorable entropychanges, DSZ55(G15) cal/deg-mol determinedhere for dimerization of the aIIbb3–rEch (1-49)M28L complex, and DSZ62(G16) cal/deg-mol forrEch (1-40 M28L) are comparable to the parameterswe have determined for the pharmaceutical integ-rin antagonists, tirofiban (66 cal/deg-mol) andeptifibatide (85 cal/deg-mol).13 Likewise, the integ-rin dimerization enthalpy changes were all com-parably unfavorable for each ligand: rEch (1-49)M28L (9 kcal/mol), rEch (1–40) M28L (12 kcal/mol), tirofiban (13 kcal/mol), eptifibatide (19 kcal/mol).13 As illustrated in Figure 5, the similar patternof enthalpy/entropy compensation for protein,ligand-mimetic, and peptide ligands supports thatconcept that a common ligand-induced oligomeri-zation mechanism is at work. Differences in themagnitude of the initial conformational pertur-bation may explain the order of effectiveness ofthese ligands at promoting oligomerization, asevidenced by the magnitude of the followingDGdimerization terms: rEch (1–49) M28L (K6.9 kcal/mol)OrEch (1-40) M28L (K6.7 kcal/mol)Oeptifiba-tide (K6.3 kcal/mol)Otirofiban (K6.1 kcal/mol).13,32

Data obtained in this study support the conceptthat the ligand-free integrin is more sensitive tothermal aggregation than the receptor–ligand com-plex. Here we have used dynamic light-scattering todetect increased integrin aggregation with a TmZ46 8C, comparable to TmZ43(G3) 8C we deter-mined by circular dichroism and intrinsic fluor-escence measurements.13 We have alsodemonstrated that rEch (1–49) M28L bindingprotects the aIIbb3 complex from thermal aggrega-tion, shifting the transition mid-point to 69 8C; thetruncation mutant rEch (1–40) M28L was somewhatless effective, yielding a maximum Tm value of59 8C. These observations extend the concept ofligand-linked thermal protection from syntheticpeptides13 to a protein ligand. While these tran-sitions are clearly outside the physiological tem-perature range, they do provide additionalbiophysical evidence that the conformational stateof the ligand-occupied integrin differs significantlyfrom its resting counterpart. Others have reportedthat echistatin binds to resting and activated aIIbb3integrins with comparable affinity,33 yet we havedemonstrated that echistatin binding is linked tochanges in aIIbb3’s tertiary and quaternary struc-ture. Our observations suggest that echistatinshifts equilibrium amongst resting, activated andclustered integrins toward a thermodynamicallystable oligomeric state.

This conformational change issue remains excit-ing and controversial, as the only available crystal-lographic data on a b3 integrin show only smallectodomain perturbations between the free and


bound avb3 complex.5,6 In contrast, biophysicalprobes and electron microscopy have detectedrather more substantial changes in tertiary structurefor both aIIbb3 and avb3, upon binding syntheticligands.8–10,12 Furthermore, SAXS measurements21

and electron microscopy26 have yielded somewhatdivergent views of the conformational pertur-bations in a5b1’s ectodomain that occur uponbinding fibronectin fragments. We anticipate thatthe lessons learned from echistatin, a model proteinligand, can serve as a paradigm for probing andunderstanding the structural prerequisites foraIIbb3’s interactions with larger adhesivemacromolecules.

Materials and Methods

aIIbb3 purification

Milligram quantities of highly purified aIIbb3 wereisolated from outdated human blood platelets (AmericanRed Cross, Triad Blood Center, Winston-Salem, NC) asdescribed.10,11 Biophysical measurements were per-formed on peak integrin fractions obtained by sizeexclusion chromatography at 4 8C on a 0.9 cm ! 85 cmcolumn of Sephacryl S-300 equilibrated in a pH 7.4 buffer(HSC-OG) containing 0.13 mol/l NaCl, 0.01 mol/l Hepes,0.002 mol/l CaCl2, 3!10K8 mol/l basic trypsin inhibitor,10K6 mol/l leupeptin, 0.02% (w/v) sodium azide, and0.03 mol/l n-octyl-b-D-glucopyranoside. Peak fractionswere then concentrated in an Amicon pressure concen-trator with a PLHK cellulose membrane, 100,000 Daretention limit.12 While we do not employ an RGD-affinity column to select for active integrin,66 we havepreviously demonstrated that our purified integrin canbind fibrinogen following transient exposure to a ligand-mimetic peptide.67 Thus our “resting yet responsive”preparation resembles the resting AS1 populationdescribed by Yan et al.;66 however, it can be readilyactivated to the AS2 state.

Cloning, expression, and purification of recombinantechistatin variants

This study employed a plasmid constructed from thevector pGEX-KG, which contains the glutathione-S-transferase (GST) gene and a thrombin-sensitive linker.68

Selected echistatin genes were then inserted at a BamHIrestriction site that added four N-terminal amino acidresidues (Gly, Ser, Thr, Met) to each disintegrin protein.Vectors expressing full-length echistatin with an M28Lmutation, rEch (1–49) M28L, and an inactive truncationvariant rEch (1–40) M28L, which is missing the C-termi-nal sequence Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr,were used here.38 Plasmid DNA was amplified bytransforming Escherichia coli strain HB101-1424471, andsequences of the resultant clones were verified (DNASequencing Core Laboratory of the ComprehensiveCancer Center of Wake Forest University School ofMedicine). rEch (1-49) M28L and rEch (1-40) M28L wereexpressed in E. coli BL21, then isolated from cell lysates byadsorption to glutathione-agarose followed by thrombin-cleavage of the bead-bound constructs.68

Each disintegrin was then purified by size-exclusionchromatography on Sephadex G-75 in 0.05 M ammoniumacetate buffer (pH 7). Elution was monitored by

absorbance at 276 nm; peak fractions were combined,lyophilized and stored at K20 8C. Purity of all recombi-nant proteins was assessed by SDS-PAGE; concentrationswere determined by quantitative amino acid compositionanalysis.10 UV–visible spectral measurements performedin a Beckman diode array spectrophotometer withrecombinant echistatin samples contained in 1 cm pathlength/0.1 ml volume quartz cuvettes providedadditional characterizations, as each disintegrin constructcontains a single tyrosine residue.38 rEch (1-49) M28Lexhibited 3276Z2300(G490) l/mol-cm (after correctionfor turbidity), in agreement with the calculated value of2030 l/mol-cm. Turbidity problems were more pro-nounced with rEch (1-40) M28L, which exhibited 3276Z3120 (G730) l/mol-cm.Mass spectrometry data obtained by MALDI-TOF

(performed by the Biomolecular Resource Facility ofWake Forest University) yielded a molecular mass of5782 Da for rEch (1-49) M28L and 4815 daltons for rEch(1-40) M28L, in reasonable agreement with the calculatedaverage masses of 5775 and 4816 Da for the full-lengthand truncated disintegrins, respectively (each as fullydisulfide-bonded proteins). Mass spectrometry dataobtained by ESIMS (Mass Spectrometry Facility of WakeForest University) yielded a monoisotopic mass of5773 Da for rEch (1-49) M28L, and peaks at 4816 and4813 Da for rEch (1-40) M28L. The calculated mono-isotopic masses for rEch (1-49) M28L range from 5770.5(four disulfide bonds) to 5778.5 (eight cysteine residues);for rEch (1-40) M28L, the calculated monoisotopic massesrange from 4812–4820 Da.

Platelet function measurements

Platelet-rich plasma (PRP) and gel-filtered platelets(GFP) were isolated from blood obtained by venipuncturefrom healthy, adult, volunteer donors as described.69

Platelet counts were determined with a Coulter MDII CellCounter (Beckman-Coulter, Miami, FL). Platelet aggrega-tion profiles were obtained in a Chrono-Log model 500aggregometer. Platelet adhesion to fibrin was determinedin a mictotiter plate adhesion assay with colorimetricread-out as described.70 This assay used highly purifiedhuman fibrinogen (free of plasminogen and factor XIII),purchased from American Diagnostica (Greenwich, CT),and highly purified human a-thrombin, purchased fromSigma Chemical Co. (St. Louis, MO).

Transmission electron microscopy

aIIbb3 samples at w1 mg/ml in HSC-OG (in thepresence/absence of 17 umol/l rEch (1-49 M28L), wereincubated for one hour at room temperature, then dilutedto w20–25 mg/ml in a buffer containing 0.05 mol/lammonium formate (pH 7.4), 30 mmol/l octyl glucoside,and 15% (v/v) glycerol. Samples were then sprayed ontofreshly cleaved mica and shadowed with tungsten in avacuum evaporator (Denton Vacuum Co., Cherry Hill,NJ).71–73 These samples were examined in a Philips 400electron microscope (FEI Co., Hillsboro, OR) operating at60 kV and a magnification of 60,000!. Size distributionmeasurements (SigmaScan, Jandel Scientific) were madefrom prints of the micrographs, using images from manydifferent areas of several different preparations to get arandom sample.

Analytical ultracentrifugation measurements

Sedimentation velocity and equilibrium measurements


were performed in a Beckman Optima XL-A analyticalultracentrifuge (Beckman Instruments, Palo Alto, CA)equipped with absorbance optics and an An60 Ti rotor.10,12

Sedimentation velocity data were collected with theaIIbb3 complex alone and in the presence of recombinantechistatin variants at 20 8C at a rotor speed of 35,000 rpm.Sedimentation velocity data were analyzed using bothSVEDBERG (version 1.04) and DCDTC (version 6.31)software (J. Philo, Thousand Oaks, CA) to obtain theweight-average sedimentation coefficient (Sw) and distri-bution of sedimenting species, g(s*), respectively.47 Allsedimentation coefficients have been corrected for solventdensity and viscosity to obtain s20,w values.The absorbance versus radial distance data obtained by

sedimentation equilibrium were analyzed by non-linearregression with WinNONLIN353 to obtain weight-average molecular weights (Mw) for the aIIbb3 complexalone and in the presence of rEch (1-49) M28L or rEch(1–40) M28L. These data were collected at temperaturesfrom 20 8C–40 8C at a rotor speed of 7000 rpm. Followingprocedures previously detailed,13 data obtained with theintegrin:echistatin complexes were subjected toadditional analyses with WinNONLIN3 to obtain a setof temperature-dependent self-association constants.74

WinNONLIN3 was provided by Dr David Yphantis andthe staff at the National Analytical UltracentrifugationFacility, Storrs, CT.

Light-scattering measurements

Static and dynamic light-scattering measurements wereperformed with the aIIbb3 complex alone and in thepresence of rEch (1-49) M28L or rEch (1–40) M28L in aMalvern Instruments ZEN 1600. Data were collected as afunction of temperature from 20 8C K90 8C, in 2.5 deg. Cincrements; each temperature ramping sequence requiredwthree hours to complete. The resultant autocorrelationfunction data were analyzed by the method of cumulantsto obtain a z-average particle diameter at each tempera-ture. Size distribution information was also obtainedfrom these corrected autocorrelation functions with theCONTIN algorithm.75

Computing the hydrodynamic parameters for integrin:echistatin complexes

Models of the aIIbb3:echistatin complex were obtainedby docking an echistatin molecule (2ECH; pdb) to atruncated version of our resting integrin model13 contain-ing aIIb residues 1–452 and b3 residues 55–432. Followingan initial manual alignment†48 echistatin–integrin con-tacts were optimized by limited energy minimizationusing CNS76. The resultant echistatin–integrin ecto-domain construct was then fused to our full-lengthmodels of either the V-shaped,5 partially bent,9

extended13 or “open” integrins.8 Hydrodynamic para-meters for these models were computed with SOMO(SOlution MOdeler) and BEAMS.48–50

Acknowledgements

We thank Dr Mark O. Lively, Director of the

† The PyMOL Molecular Graphics System DeLanoScientific, San Carlos, CA (2002).

Biomolecular Resource Facility of Wake ForestUniversity, and Dr Michael J. Thomas, Director ofthe Mass Spectrometry Facility of Wake ForestUniversity, as well as Mark Morris and MikeSamuel, for mass determinations. We thank DrJulie Edelson for her expert editorial assistance.

This investigation was supported by Grant-in-Aid 0355869U from the American Heart Associationand the Venture Fund of Wake Forest UniversitySchool of Medicine (to R.R.H.), National Institutesof Health Postdoctoral Fellowship F32-AI051805 (toJ.H.C.), National Institutes of Health grant AI15892(to D.S.L.), European Community grant GI04-CT96-0662 (to M.R.), and National Institutes of Healthgrant HL 30954 (to J.W.W.).

References

1. Hynes, R. O. (2002). Integrins: bidirectional, allostericsignaling machines. Cell, 110, 673–687.

2. Xiong, J. P., Stehle, T., Goodman, S. L. & Arnaout,M. A. (2003). New insights into the structural basisof integrin activation. Blood, 102, 1155–1159.

3. Humphries, M. J., McEwan, P. A., Barton, S. J.,Buckley, P. A., Bella, J. & Paul, M. A. (2003). Integrinstructure: heady advances in ligand binding, butactivation still makes the knees wobble. TrendsBiochem. Sci. 28, 313–320.

4. Carman, C. V. & Springer, T. A. (2003). Integrin avidityregulation: are changes in affinity and conformationunderemphasized? Curr. Opin. Cell Biol, 15, 547–556.

5. Xiong, J. P., Stehle, T., Diefenbach, B., Zhang, R.,Dunker, R., Scott, D. L. et al. (2001). Crystal structureof the extracellular segment of integrin aVb3. Science,294, 339–345.

6. Xiong, J. P., Stehle, T., Zhang, R., Joachimiak, A.,Frech, M., Goodman, S. L. & Arnaout, M. A. (2002).Crystal structure of the extracellular segment ofintegrin aVb3 in complex with an Arg-Gly-Aspligand. Science, 296, 151–155.

7. Weisel, J. W., Nagaswami, C., Vilaire, G. & Bennett,J. S. (1992). Examination of the platelet membraneglycoprotein IIb–IIIa complex and its interactionwith fibrinogen and other ligands by electronmicroscopy. J. Biol. Chem. 267, 16637–16643.

8. Takagi, J., Petre, B. M., Walz, T. & Springer, T. A.(2002). Global conformational rearrangements inintegrin extracellular domains in outside–in andinside–out signaling. Cell, 110, 599–611.

9. Adair, B. D. & Yeager, M. (2002). Three-dimensionalmodel of the human platelet integrin aIIbb3 based onelectron cryomicroscopy and X-ray crystallography.Proc. Natl Acad. Sci. USA, 99, 14059–14064.

10. Hantgan, R. R., Paumi, C., Rocco, M. & Weisel, J. W.(1999). Effects of ligand-mimetic peptides Arg-Gly-Asp-X (XZPhe, Trp, Ser) on aIIbb3 integrin con-formation and oligomerization. Biochemistry, 38,14461–14464.

11. Hantgan, R. R., Braaten, J. V. & Rocco, M. (1993).Dynamic light scattering studies of aIIbb3 solutionconformation. Biochemistry, 32, 3935–3941.

12. Hantgan, R. R., Rocco, M., Nagaswami, C. & Weisel,J. W. (2001). Binding of a fibrinogen mimeticstabilizes integrin aIIbb3’s open conformation.Protein Sci. 10, 1614–1626.

13. Hantgan, R. R., Lyles, D. S., Mallett, T. C., Rocco, M.,


Nagaswami, C. &Weisel, J. W. (2003). Ligand bindingpromotes the entropy-driven oligomerization ofintegrin aIIbb3. J. Biol. Chem. 278, 3417–3426.

14. Liddington, R. C. (2002). Will the real integrin pleasestand up? Structure, 10, 605–607.

15. Arnaout, M. A., Goodman, S. L. & Xiong, J.-P. (2002).Coming to grips with integrin binding to ligands.Curr. Opin. Cell Biol. 14, 641–651.

16. Weisel, J. W., Nagaswami, C., Vilaire, G. & Bennett,J. S. (1992). Examination of the platelet membraneglycoprotein IIb–IIIa complex and its interactionwith fibrinogen and other ligands by electronmicroscopy. J. Biol. Chem. 267, 16637–16643.

17. Nermut, M. V., Green, N. M., Eason, P., Yamada, S. S.& Yamada, K. M. (1988). Electron microscopy andstructural model of human fibronectin receptor.EMBO J. 7, 4093–4099.

18. Carrell, N. A., Fitzgerald, L. A., Steiner, B., Erickson,H. P. & Phillips, D. R. (1985). Structure of humanplatelet glycoproteins IIb and IIIa as determined byelectron microscopy. J. Biol. Chem. 260, 1743–1749.

19. Xiong, J. P., Stehle, T., Goodman, S. L. & Arnaout,M. A. (2003). Integrins, cations and ligands: makingthe connection. J. Thromb. Haemost. 1, 1642–1654.

20. Liddington, R. C. & Ginsberg, M. H. (2002). Integrinactivation takes shape. J. Cell Biol. 158, 833–839.

21. Mould, A. P., Symonds, E. J., Buckley, P. A.,Grossmann, J. G., McEwan, P. A., Barton, S. J. et al.(2003). Structure of an integrin-ligand complexdeduced from solution X-ray scattering and site-directed mutagenesis. J. Biol. Chem. 278, 39993–39999.

22. Takagi, J. & Springer, T. A. (2002). Integrin activationand structural rearrangement. Immunol. Rev. 186,141–163.

23. Beglova, N., Blacklow, S. C., Takagi, J., Springer, T. A.(2002). Cysteine-rich module structure reveals afulcrum for integrin rearrangement upon activation.Nature Struct. Biol. 9, 282–287.

24. Luo, B. H., Springer, T. A. & Takagi, J. (2003).Stabilizing the open conformation of the integrinheadpiece with a glycan wedge increases affinity forligand. Proc. Natl Acad. Sci. USA, 100, 2403–2408.

25. Luo, B. H., Springer, T. A. & Takagi, J. (2003). Highaffinity ligand binding by integrins does not involvehead separation. J. Biol. Chem. 278, 17185–17189.

26. Takagi, J., Strokovich, K., Springer, T. A. & Walz, T.(2003). Structure of integrin a5b1 in complex withfibronectin. EMBO J. 22, 4607–4615.

27. Kim, M., Carman, C. V. & Springer, T. A. (2003).Bidirectional transmembrane signaling by cyto-plasmic domain separation in integrins. Science, 301,1720–1725.

28. Gan, Z. R., Gould, R. J., Jacobs, J. W., Friedman, P. A. &Polokoff, M. A. (1988). Echistatin. A potent plateletaggregation inhibitor from the venom of the viper,Echis carinatus. J. Biol. Chem. 263, 19827–19832.

29. Saudek, V., Atkinson, R. A. & Pelton, J. T. (1991).Three-dimensional structure of echistatin, the smal-lest active RGD protein. Biochemistry, 30, 7369–7372.

30. Atkinson, R. A., Saudek, V. & Pelton, J. T. (1994).Echistatin: the refined structure of a disintegrin insolution by 1H NMR and restrained moleculardynamics. Int. J. Pept. Protein Res. 43, 563–572.

31. Chen, Y., Suri, A. K., Kominos, D., Sanyal, G., Naylor,A. M., Pitzenberger, S. M. et al. (1994). Three-dimensional structure of echistatin and dynamics ofthe active site. J. Biomol. NMR, 4, 307–324.

32. Hantgan, R. R., Stahle, M. C., Jerome, W. G.,Nagaswami, C. & Weisel, J. W. (2002). Tirofiban

blocks platelet adhesion to fibrin with minimalperturbatin of GpIIb/IIIa structure. Thromb. Haemost.87, 910–917.

33. Bednar, R. A., Gaul, S. L., Hamill, T. G., Egbertson,M. S., Shafer, J. A., Hartman, G. D. et al. (1998).Identification of low molecular weight gp iib/iiiaantagonists that bind preferentially to activatedplatelets. J. Pharmacol. Expt. Ther. 285, 1317–1326.

34. Shattil, S. J. (1999). Signaling through platelet integrinaIIbb3: inside–out, outside–in, and sideways. Thromb.Haemost. 82, 318–325.

35. Tomiyama, Y., Tsubakio, T., Piotrowicz, R. S., Kurata,Y., Loftus, J. C. & Kunicki, T. J. (1992). The Arg-Gly-Asp (RGD) recognition site of platelet glycoproteinIIb–IIIa on nonactivated platelets is accessible to high-affinity macromolecules. Blood, 79, 2303–2312.

36. D’Souza, S. E., Ginsberg, M. H. & Plow, E. F. (1991).Arginyl-glycyl-aspartic acid (RGD): a cell adhesionmotif. Trends Biochem. Sci. 16, 246–250.

37. Marcinkiewicz, C., Vijay-Kumar, S., McLane, M. A. &Niewiarowski, S. (1997). Significance of RGD loopand C-terminal domain of echistatin for recognition ofaIIbb3 and avb3 integrins and expression of ligand-induced binding site. Blood, 90, 1565–1575.

38. Wierzbicka-Patynowski, I., Niewiarowski, S.,Marcinkiewicz, C., Calvete, J. J., Marcinkiewicz, M. M.& McLane, M. A. (1999). Structural requirements ofechistatin for the recognition of aVb3 and a5b1integrins. J. Biol. Chem. 274, 37809–37814.

39. Scheibler, L., Mierke, D. F., Bitan, G., Rosenblatt, M. &Chorev, M. (2001). Identification of a contact domainbetween echistatin and the integrin aVb3 by photo-affinity cross-linking. Biochemistry, 40, 15117–15126.

40. Yahalom, D., Wittelsberger, A., Mierke, D. F.,Rosenblatt, M., Alexander, J. M. & Chorev, M.(2002). Identification of the principal binding site forRGD-containing ligands in the aVb3 integrin: aphotoaffinity cross-linking study. Biochemistry, 41,8321–8331.

41. Shimaoka, M., Lu, C., Salas, A., Xiao, T., Takagi, J. &Springer, T. A. (2002). Stabilizing the integrin alpha Minserted domain in alternative conformations with arange of engineered disulfide bonds. Proc. Natl Acad.Sci. USA, 99, 16737–16741.

42. Hantgan, R. R. (1988). Fibrin protofibril and fibrino-gen binding to ADP-stimulated platelets: evidencefor a common mechanism. Biochim. Biophys. Acta, 968,24–35.

43. Hantgan, R. R., Taylor, R. G. & Lewis, J. C. (1985).Platelets interact with fibrin only after activation.Blood, 65, 1299–1311.

44. Li, R., Mitra, N., Gratkowski, H., Vilaire, G., Litvinov,R., Nagasami, C., Weisel, J. W. et al. (2003). Activationof integrin aIIbb3 by modulation of transmembranehelix associations. Science, 300, 795–798.

45. Edwards, A. L. (1979). Multiple Regression and theAnalysis of Variance and Covariance. W. H. Freeman andCompany, San Francisco pp. 1–212.

46. Koradi, R., Billeter, M. & Wuthrich, K. (1996).MOLMOL: a program for display and analysis ofmacromolecular structures. J. Mol. Graph. 14, 51–55.

47. Stafford, W. F., III (1992). Boundary analysis insedimentation transport experiments: a procedurefor obtaining sedimentation coefficient distributionsusing the time derivative of the concentration profile.Anal. Biochem. 203, 295–301.

48. Spotorno, B., Piccinini, L., Tassara, G., Ruggiero, C.,Nardini, M., Molina, F. & Rocco, M. (1997). BEAMS(beads modeling system): a set of computer programs


for the visualization and the computation of hydro-dynamic and conformational properties of beadsmodels of proteins. Eur. Biophys. J. 25, 373–384.

49. Ray, N. (2001). The development of experimental andcomputational tools for studying protein interactions:applied to the bacterial toxin pneumolysin. PhDThesis, University of Glasgow, UK.

50. Byron, O. (2000). Hydrodynamic bead modeling ofbiological macromolecules. Methods Enzymol. 321,278–304.

51. Buensuceso, C., De Virgilio, M. & Shattil, S. J. (2003).Detection of integrin aIIbb3 clustering in living cells.J. Biol. Chem. 278, 15217–15224.

52. Hantgan, R. R., Lyles, D. S., Rocco, M. & Weisel, J. W.(2002). Ligation effects the thermal stability of integrinaIIbb3. Biophys. J. 82, 38.

53. Johnson, M., Correia, J. J., Yphantis, D. A. &Halvorson, H. (1981). Analysis of data from theanalytical ultracentrifuge by nonlinear least-squaretechniques. Biophys. J. 36, 575–588.

54. Arnaout, M. A. (2002). Integrin structure: new twistsand turns in dynamic cell adhesion. Immunol. Rev. 186,125–140.

55. Shimaoka, M., Takagi, J. & Springer, T. A. (1931).Conformational regulation of integrin structure andfunction. Annu. Rev. Biophys. Biomol. Struct. 2002,485–516.

56. Rivas, G. A., Aznarez, J. A., Usobiaga, P., Salz, J. L. &Gonzalez-Rodriguez, J. (1991). Molecular characteriz-ation of the human platelet integrin gpIIb/IIIa and itsconstituent glycoproteins. Eur. Biophys. J. 19, 335–345.

57. Rocco, M., Spotorno, B. & Hantgan, R. R. (1993).Modeling the aIIbb3 integrin solution conformation.Protein Sci. 2, 2154–2166.

58. Creigton, T. E. (1993). Enzyme catalysis. In Proteins:Structures and Molecular Properties (Creigton, T. E., ed),pp. 385–461,W. H. Freeman and Company, NewYork.

59. Feuston, B. P., Culberson, J. C., Duggan, M. E.,Hartman, G. D., Leu, C. T. & Rodan, S. B. (2002).Binding model for nonpeptide antagonists of aVb3integrin. J. Med. Chem. 45, 5640–5648.

60. Gottschalk, K. E., Adams, P. D., Brunger, A. T. &Kessler, H. (2002). Transmembrane signal transduc-tion of the aIIbb3 integrin. Protein Sci. 11, 1800–1812.

61. Fleming, K. G. (2002). Standardizing the free energychange of transmembrane helix-helix interactions.J. Mol. Biol. 323, 563–571.

62. Gottschalk, K. E. & Kessler, H. (2004). Evidence forhetero-association of transmembrane helices ofintegrins. FEBS Letters, 557, 253–258.

63. Kumar, M. S., Carson, M., Hussain, M. M. & Murthy,H. M. (2002). Structures of apolipoprotein A-II and alipid-surrogate complex provide insights into apoli-poprotein–lipid interactions. Biochemistry, 41,11681–11691.

64. Shu, W., Ji, H. & Lu, M. (2000). Interactions between

HIV-1 gp41 core and detergents and their impli-cations for membrane fusion. J. Biol. Chem. 275,1839–1845.

65. Roth, M., Ducruix, A. & Husson-Reiss, F. (1991).Structure of the detergent phase and protein-deter-gent interactions in crystals of the wild-type (Strain Y)Rhodobacter sphaeroides photochemical reaction center.Biochemistry, 30, 9403–9413.

66. Yan, B., Hu, D. D., Knowles, S. K. & Smith, J. W. (2000).Probing chemical and conformational differences inthe resting and active conformers of platelet integrinaIIbb3. J. Biol. Chem. 275, 7249–7260.

67. Bonnefoy, A., Hantgan, R., Legrand, C. & Frojmovic,M. M. (2001). A model of platelet aggregationinvolving multiple interactions of thrombospondin-1, fibrinogen, and GPIIbIIIa receptor. J. Biol. Chem.276, 5605–5612.

68. Smith, D. B. & Johnson, K. S. (1988). Single-steppurification of polypeptides expressed in Escherichiacoli as fusions with glutathione S-transferase. Gene, 67,31–40.

69. Hantgan, R. R. (1984). A study of the kinetics of ADP-triggered platelet shape change. Blood, 64, 896–906.

70. Hantgan, R. R., Stahle, M., Del, G. V., Adams, M.,Lasher, T., Jerome, W. G. et al. (2001). aIIb’s cyto-plasmic domain is not required for ligand-inducedclustering of integrin aIIbb3. Biochim. Biophys. Acta,1540, 82–95.

71. Fowler, W. E. & Erickson, H. P. (1979). Trinodularstructure of fibrinogen. Confirmation by bothshadowing and negative stain electron microscopy.J. Mol. Biol. 134, 241–249.

72. Veklich, Y. I., Gorkun, O. V., Medved, L. V.,Nieuwenhuizen, W. & Weisel, J. W. (1993). Carboxyl-terminal portions of the a chains of fibrinogen andfibrin. Localization by electron microscopy and theeffects of isolated aC fragments on polymerization.J. Biol. Chem. 268, 13577–13585.

73. Weisel, J. W., Stauffacher, C. V., Bullitt, E. & Cohen, C.(1985). A model for fibrinogen: domains andsequence. Science, 230, 1388–1391.

74. Laue, T. M. (1995). Sedimentation equilibrium as athermodyamic tool. Methods Enzymol. 259, 427–452.

75. Provencher, S. W. (1982). CONTIN: a general purposeconstrained regularization program for invertingnoisy linear algebraic and integral equations. Comp.Phys. Commun. 27, 229–242.

76. Brunger, A. T., Adams, P. D., Clore, G. M.,DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W.et al. (1998). Crystallography & NMR system: a newsoftware suite for macromolecular structure determi-nation. Acta Crystallog. sect. D, 54, 905–921.

77. Philo, J. S. (1997). An improved function for fittingsedimentation velocity data for low-molecular-weight solutes. Biophys. J. 72, 435–444.

Edited by W. Baumeister

(Received 4 June 2004; received in revised form 3 August 2004; accepted 4 August 2004)

The Disintegrin Echistatin Stabilizes Integrin αIIbβ3's Open Conformation and Promotes Its...

Documents

Transcript of The Disintegrin Echistatin Stabilizes Integrin αIIbβ3's Open Conformation and Promotes Its...