Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.     

Role of arginine-38 in regulation of the cytochrome c oxidation-reduction equilibrium

Arg-38 is an internal residue of mitochondrial cytochrome c that is close to heme propionate-7. Previous work comparing the behavior of cytochromes c from several species [Moore, G. R., Harris, D. E., Leitch, F. A., & Pettigrew, G. W. (1984) Biochim. Biophys. Acta 764, 331-342] has suggested that Arg-38 lowers the pKa of this propionate group and thereby accounts for the relative pH independence of the cytochrome c reduction potential from pH 5 to pH 8. The influence of Arg-38 on the oxidation-reduction equilibrium of yeast iso-1-cytochrome c has now been investigated by electrochemical, NMR, and theoretical analysis of six specifically mutated forms of this protein in which Arg has been replaced by Lys, His, Gln, Asn, Leu, or Ala. As the electron-withdrawing character of the residue at position 38 decreases, the reduction potential of the protein also decreases, with the largest decrease (ca. 50 mV) observed for the Ala variant. However, the variation in the reduction potentials of the mutants as a function of pH was similar to that observed for the wild-type protein. The effects of some of these mutations on the pKa values of His-33 and His-39 have been determined by NMR spectroscopy and found to be minimal. Calculations of the electrostatic free energy for the Leu-38 variant predict a decrease in the reduction potential of this mutant that is remarkably close to that observed experimentally. This work establishes that while Arg-38 contributes to the relatively high reduction potential of cytochrome c, this residue does not appear to be the sole functionality responsible for lowering the heme propionate-7 pKa.     

Role of arginine-43 and arginine-69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites

The Hin recombinase mediates the site-specific inversion of a segment of the Salmonella chromosome between two flanking 26 bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4 bp added to the centre of the site, unlike wild-type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA-binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.     

Role of glutamate-126 and arginine-144 in the lactose permease of Escherichia coli

Several previous studies have suggested that glutamate-126 and arginine-144 in the lactose permease of Escherichia coli form an ion pair that is essential for sugar binding. To further investigate the role of these residues, E126Q, R144Q, and R144S mutants were made. The R144Q and R144S strains, which had negligible levels of transport, were used as parental strains to isolate suppressor mutations that partially restored sugar transport. The R144Q parent only yielded first-site revertants, but the R144S strain produced three types of second-site replacements: E126Q, V229A, and L330R. In downhill transport assays, the E126Q strain was able to transport lactose at low levels, with an apparent K(m) 3-fold higher than the wild-type strain but a severely depressed apparent V(max). A triple mutant, E126Q/R144S/V229A, showed a relatively robust V(max) value for downhill transport and could actively accumulate lactose against a concentration gradient. Taken together, these results indicate that Glu-126 and Arg-144 are not essential for sugar binding. An alternative explanation for their role in maintaining secondary structure is discussed.     

Phosphorylation-induced change of the oligomerization state of alpha B-crystallin

alphaB-crystallin in cells can be phosphorylated at three serine residues in response to stress or during mitosis (Ito, H., Okamoto, K., Nakayama, H., Isobe, T., and Kato, K. (1997) J. Biol. Chem. 272, 29934-29941 and Kato, K., Ito, H., Kamei, K., Inaguma, Y., Iwamoto, I., and Saga, S. (1998) J. Biol. Chem. 273, 28346-28354). In the present study, we determined effects of phosphorylation of alphaB-crystallin on its oligomerization state, mainly by using site-directed mutagenesis, in which all three phosphorylation sites were substituted with aspartate to mimic the phosphorylation state (3D-alphaB). From results of sucrose density gradient centrifugation, we found that wild type alphaB-crystallin (wt-alphaB) and 3D-alphaB sedimented in fractions corresponding to apparent molecular masses of about 500 and 300 kDa, respectively. Chaperone-like activity of 3D-alphaB was significantly weaker than that of wt-alphaB. When wt-alphaB and 3D-alphaB were expressed in COS-m6 cells, they sedimented at positions corresponding to apparent molecular masses of about 500 and 100 kDa, respectively. In U373 MG human glioma cells, alphaB-crystallin was observed as large oligomers with apparent molecular masses about 500 kDa and the oligomerization size was reduced after phosphorylation induced by phorbol 12-myristate 13-acetate and okadaic acid. Coexpression of luciferase and wt-alphaB or 3D-alphaB in Chinese hamster ovary cells caused protection of the enzyme from heat inactivation although the degree of protection with 3D-alphaB was less than that with wt-alphaB. From these observations, it is suggested that phosphorylation of alphaB-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the case of HSP27.     

Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum

In contrast to most multimeric transmembrane complexes that oligomerize in the endoplasmic reticulum (ER), the gap junction protein connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus. The mechanisms that prevent oligomerization of Cx43 and related connexins in the ER are not well understood. Also, some studies suggest that connexins can oligomerize in the ER. We used connexin constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, Cx43-HKKSL was retained in the ER and prevented from oligomerization. However, another ER-retained HKKSL-tagged connexin, Cx32-HKKSL, had the capacity to oligomerize. Because this suggested that Cx43 contains a motif that prevented oligomerization in the ER, a series of HKKSL-tagged and untagged Cx32/Cx43 chimeras was screened to define this motif. The minimal motif, which prevented ER oligomerization, consisted of the complete third transmembrane domain and the second extracellular loop from Cx43 on a Cx32 backbone. We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer.     

Role of the amino terminal domain in GroES oligomerization

Digestions of the GroES oligomer with trypsin, chymotrypsin and Glu-C protease from Staphylococcus aureus V8 (V8) have helped to locate three regions in the GroES sequence that are sensitive to limited proteolysis and have provided information of the GroES domains involved in monomer-monomer and GroEL interaction. The removal of the first 20 or 27 amino acids of the N-terminal region of each GroES monomer by trypsin or chymotrypsin respectively, abolish the oligomerization of the GroES complex and its binding to GroEL. The V8-treatment of GroES promotes the breakage of the peptide bond between Glu₁₈ and Thr₁₉ but not the liberation of the N-terminal fragment from the GroES oligomer, which is capable of forming with GroEL a complex active in protein folding. It is deduced from these results that the N-terminal region of the GroES monomer is involved in monomer-monomer interaction, providing experimental evidence that relates some biochemical properties of GroES with its three-dimensional structure at atomic resolution.     

Role of Arg163 in the N-glycosidase activity of neo-trichosanthin

Three mutant crystals of neo-trichosanthin (n-TCS), R163K, R163H and R163Q, were obtained by the hanging drop vapor diffusion method. Structure determination indicated that there are no significant differences between the mutants and n-TCS except in the active pocket. All of them were also soaked in sodium citrate buffer (pH 4. 5) containing 20% KCl and 10 mg/ml AMP. Structure determination suggests that in the active pocket of the crystals of R163K and R163H, parallel to the aromatic ring of Tyr70, each mutant possesses an adenine. The relationship between structure and function is discussed. Biochemical analysis reveals that the mutants R163K and R163H have N-glycosidase activity, while R163Q does not. This suggests that R163 is a crucial residue for the enzyme activity of n-TCS, and its role is providing proton.     

Influence of the C-terminal residues on oligomerization of alpha A-crystallin

Earlier studies have shown that the chaperone activity of alpha-crystallin is significantly affected in diabetic rat and human lenses. Subsequently, mass spectrometric analysis showed diabetic lenses having high levels of the alphaA-crystallins in which different numbers of C-terminal residues were deleted. The present study was aimed to show whether cleavage of these residues influences protein structure, oligomerization, and chaperone function. For generation of various mutants, a stop codon was introduced at the positions of interest, proteins were expressed in BL21(DE3)pLys S E. coli, and the truncated alphaA-crystallins were purified by size-exclusion chromatography. The molecular masses, as determined by molecular sieve HPLC, of mutants with deletions of 1, 5, and 10 C-terminal residues (group-1) were 519-602 kDa, and those of mutants with deletions of 11, 16, and 22 C-terminal residues (group-2) were 148-152 kDa, as compared to 607 kDa for alphaA-wild type. On the basis of circular dichroism measurements, the alpha helix content was 2-fold higher and the tertiary structure was significantly altered in the group-2 mutants. Chaperoning abilities, as determined by the ADH assay and the betaL-crystallin heat denaturation assay, of the group-1 mutants, with the exception of alphaA(1-163), were slightly improved or unchanged, that of alphaA(1-163) was moderately affected, and those of the group-2 mutants were severely affected. Most strikingly, cleavage of 11 C-terminal residues including Arg-163 showed a substantial decrease in oligomeric size and chaperone function and significant changes in protein structure whereas cleavage of 10 residues had either a small effect or no effect at all. This points to an important role for the C-terminal extension, Arg-163 in particular, and no significant role for the C-terminal flexible tail in the oligomer assembly of alphaA-crystallin.     

Role of arginine-304 in the diphosphate-triggered active site closure mechanism of trichodiene synthase

The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PP(i)) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PP(i) bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg2+(3)-PP(i), in which R304 donates hydrogen bonds to PP(i) and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP(i) in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP(i) and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg2+(3)-PP(i) binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP(i) leaving group.     

Domain swapping in human alpha A and alpha B crystallins affects oligomerization and enhances chaperone-like activity

alphaA and alphaB crystallins, members of the small heat shock protein family, prevent aggregation of proteins by their chaperone-like activity. These two proteins, although very homologous, particularly in the C-terminal region, which contains the highly conserved "alpha-crystallin domain," show differences in their protective ability toward aggregation-prone target proteins. In order to investigate the differences between alphaA and alphaB crystallins, we engineered two chimeric proteins, alphaANBC and alphaBNAC, by swapping the N-terminal domains of alphaA and alphaB crystallins. The chimeras were cloned and expressed in Escherichia coli. The purified recombinant wild-type and chimeric proteins were characterized by fluorescence and circular dichroism spectroscopy and gel permeation chromatography to study the changes in secondary, tertiary, and quaternary structure. Circular dichroism studies show structural changes in the chimeric proteins. alphaBNAC binds more 8-anilinonaphthalene-1-sulfonic acid than the alphaANBC and the wild-type proteins, indicating increased accessible hydrophobic regions. The oligomeric state of alphaANBC is comparable to wild-type alphaB homoaggregate. However, there is a large increase in the oligomer size of the alphaBNAC chimera. Interestingly, swapping domains results in complete loss of chaperone-like activity of alphaANBC, whereas alphaBNAC shows severalfold increase in its protective ability. Our findings show the importance of the N- and C-terminal domains of alphaA and alphaB crystallins in subunit oligomerization and chaperone-like activity. Domain swapping results in an engineered protein with significantly enhanced chaperone-like activity.     

Role of Arg-166 in yeast cytochrome C1

A systematic screen for dominant-negative mutations of the CYT1 gene, which encodes cytochrome c(1), revealed seven mutants after testing approximately 10(4) Saccharomyces cerevisiae strains transformed with a library of mutagenized multicopy plasmids. DNA sequence analysis revealed multiple nucleotide substitutions with six of the seven altered Cyt1p having a common R166G replacement, either by itself or accompanied with other amino acid replacements. A single R166G replacement produced by site-directed mutagenesis demonstrated that this change produced a nearly nonfunctional cytochrome c(1), with diminished growth on glycerol medium and diminished respiration but with the normal or near normal level of cytochrome c(1) having an attached heme group. In contrast, R166K, R166M, or R166L replacements resulted in normal or near normal function. Arg-166 is conserved in all cytochromes c(1) and lies on the surface of Cyt1p in close proximity to the heme group but does not seem to interact directly with any of the physiological partners of the cytochrome bc(1) complex. Thus, the large size of the side chain at position 166 is critical for the function of cytochrome c(1) but not for its assembly in the cytochrome bc(1) complex.     

A critical motif for oligomerization and chaperone activity of bacterial alpha-heat shock proteins.

Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all alpha-crystallin type heat shock proteins (alpha-Hsp), but the molecular details of complex assembly are poorly understood. The alpha-Hsp proteins from Bradyrhizobium japonicum are suitable bacterial models for structure-function studies of these ubiquitous stress proteins. They fall into two distinct classes, A and B, display chaperone activity in vitro and form oligomers of approximately 24 subunits. We constructed 19 derivatives containing truncations or point mutations within the N- and C-terminal regions and analyzed them by gel filtration, citrate synthase assay and coaffinity purification. Truncation of more than the initial few amino acids of the N-terminal region led to the formation of distinct dimeric to octameric structures devoid of chaperone activity. In the C-terminal extension, integrity of an isoleucine-X-isoleucine (I-X-I) motif was imperative for alpha-Hsp functionality. This I-X-I motif is one of the characteristic consensus motifs of the alpha-Hsp family, and here we provide experimental evidence of its structural and functional importance. alpha-Hsp proteins lacking the C-terminal extension were inactive, but still able to form dimers. Here, we demonstrate that the central alpha-crystallin domain alone is not sufficient for dimerization. Additional residues at the end of the N-terminal region were required for the assembly of two subunits.     

Differential effects of two mutations at arginine-234 in the alpha subunit of human pyruvate dehydrogenase.

The most common mutation in the alpha subunit of the pyruvate dehydrogenase (E1) component of the human pyruvate dehydrogenase complex (PDC) is arginine-234 to glycine and glutamine in 12 and 3 patients, respectively. Interestingly, these two mutations at the same amino acid position cause E1 (and hence PDC) deficiency by apparently different mechanisms. Recombinant human R234Q E1 had similar V(max) (25.7 +/- 4.4 units/mg E1) and apparent K(m) (101 +/- 4 nM) values for TPP as recombinant wild-type human E1, while R234G E1 had no significant change in V(max) (33.6 +/- 4.7 units/mg E1) but had a 7-fold increase in its apparent K(m) value for TPP (497 +/- 25 nM). Both of the R234 mutant proteins had similar apparent K(m) values for pyruvate. Both R234Q and R234G mutant proteins displayed similar phosphorylation rates of sites 1 and 2 by pyruvate dehydrogenase kinase 2 (PDK2) and site 3 by PDK1 compared to wild-type E1. Phosphorylated R234Q E1, R234G E1, and wild-type E1 also had similar dephosphorylation rates of sites 1 and 2 by phosphopyruvate dehydrogenase phosphatase 1. The rate of dephosphorylation of site 3 was about 50% for R234Q E1 and without a significant change for R234G E1 compared to the wild type. The data indicate that the patients with the R234G E1 mutation are symptomatic due to a decreased ability of this mutant protein to bind TPP, whereas the patients with the R234Q E1 mutation are symptomatic due to a decreased rate of dephosphorylation of site 3, hence keeping the enzyme in a phosphorylated/inactivated form.     

Role of oligomerization domains in thrombospondins and other extracellular matrix proteins

Coiled coils, collagen triple helices and globular oligomerization domains mediate the subunit assembly of many proteins in vertebrates and invertebrates. Oligomerization offers functional advantages including multivalency, increased binding strength and the combined function of different domains. These features are seen in natural proteins and may be introduced by protein engineering. The special focus of this review is on oligomerization domain of extracellular matrix proteins. For thrombospondins, initial interesting results on the functional role of oligomerization have been published. Other features remain to be explored. For example, it is not clear why thrombospondin-1 and thrombospondin-2 are trimers whereas thrombospondins-3 to -5 are pentamers. To stimulate this type of research, this review makes a survey of oligomerization domains and their functional role in extracellular matrix proteins.     

Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis

X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of aspartate aminotransferase. It forms a salt bridge with the beta or gamma carboxylate group of the substrate [Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., & Christen, P. (1984) J. Mol. Biol. 174, 497-525]. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity (kcat/KM) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and alpha-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate and a number of aromatic and other neutral amino acids is reduced by only 2-9 fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration, no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (kcat/KM)cationic/(kcat/KM)anionic is in the range 2-40-fold for R292D, whereas this ratio has a range of [(0.3-6) x 10(-6)]-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the alpha-helix 163-170 in factor Xa catalytic activity

Factor Xa (FXa) is a key protease of the coagulation pathway whose activity is known to be in part modulated by binding to factor Va (FVa) and sodium ions. Previous investigations have established that solvent-exposed, charged residues of the FXa alpha-helix 163-170 (h163-170), Arg(165) and Lys(169), participate in its binding to FVa. In the present study we aimed to investigate the role of the other residues of h163-170 in the catalytic functions of the enzyme. FX derivatives were constructed in which point mutations were made or parts of h163-170 were substituted with the corresponding region of either FVIIa or FIXa. Purified FXa derivatives were compared with wild-type FXa. Kinetic studies in the absence of FVa revealed that, compared with wild-type FXa, key functional parameters (catalytic activity toward prothrombin and tripeptidyl substrates and non-enzymatic interaction of a probe with the S1 site) were diminished by mutations in the NH(2)-terminal portion of h163-170. The defective amidolytic activity of these FXa derivatives appears to result from their impaired interaction with Na(+) because using a higher Na(+) concentration partially restored normal catalytic parameters. Furthermore, kinetic measurements with tripeptidyl substrates or prothrombin indicated that assembly of these FXa derivatives with an excess of FVa in the prothrombinase complex improves their low catalytic efficiency. These data indicate that residues in the NH(2)-terminal portion of the FVa-binding h163-170 are energetically linked to the S1 site and Na(+)-binding site of the protease and that residues Val(163) and Ser(167) play a key role in this interaction.     

Role of the tyrosine corner motif in the stability of neocarzinostatin

Although the immunoglobulin-like beta-sandwich fold has no specifically conserved function, some common structural features have been observed, in particular a structural motif, the tyrosine corner. Such a motif was described in neocarzinostatin (NCS), a bacterial protein the structure of which is very similar to that of the immunoglobulin domain. Compared with the other beta-sheet proteins, the NCS 'tyrosine corner' presents non-standard structural features. To investigate the role of this motif in the NCS structure and stability, we studied the properties of a mutant where the H bond interaction had been eliminated by replacing the tyrosine with a phenylalanine. This mutation costs 4.0 kcal/mol showing that the NCS 'tyrosine corner' is involved in protein stability as in the other Greek key proteins. This destabilization is accompanied by remote structural effects, including modification of the binding properties, suggesting an increase in the internal flexibility of the protein. With a view to using this protein for drug targeting, these results along with those obtained previously allow us to define clearly the limitations of the modifications that can be performed on this scaffold.     

Ligand binding promotes the entropy-driven oligomerization of integrin alpha IIb beta 3

Integrin alpha(IIb)beta(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 alpha(IIb)beta(3). The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at approximately 43 degrees 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 gamma-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 alpha(IIb)beta(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 degrees 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 alpha(IIb)beta(3) protomers and dimers based on the ectodomain structure of alpha(v)beta(3). We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.