βA Domain Research Articles

Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation.

Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the alphaIIbbeta3 receptor. In this study we identified regions on integrin beta3 that control 7E3 binding. Murine/human amino acid substitutions were created in two regions of the betaA domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human beta3 in complex with alphaIIb. The introduction of both the human C177-C184 region and human W129 into murine beta3 was necessary and sufficient to permit 7E3 binding to the human alphaIIb/murine beta3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 A of each other in the crystal structure and close to the beta3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that alphaIIbbeta3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an alphaIIbbeta3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with alphaIIbbeta3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in alphaIIbbeta3 adopting a more extended conformation.

Crystal Structure of Trimestatin, a Disintegrin Containing a Cell Adhesion Recognition Motif RGD

Disintegrins are a family of small proteins containing an Arg-Gly-Asp (RGD) sequence motif that binds specifically to integrin receptors. Since the integrin is known to serve as the final common pathway leading to aggregation via formation of platelet–platelet bridges, disintegrins act as fibrinogen receptor antagonists. Here, we report the first crystal structure of a disintegrin, trimestatin, found in snake venom. The structure of trimestatin at 1.7 Å resolution reveals that a number of turns and loops form a rigid core stabilized by six disulfide bonds. Electron densities of the RGD sequence are visible clearly at the tip of a hairpin loop, in such a manner that the Arg and Asp side-chains point in opposite directions. A docking model using the crystal structure of integrin αVβ3 suggests that the Arg binds to the propeller domain, and Asp to the βA domain. This model indicates that the C-terminal region is another potential binding site with integrin receptors. In addition to the RGD sequence, the structural evidence of a C-terminal region (Arg66, Trp67 and Asn68) important for disintegrin activity allows understanding of the high affinity and selectiveness of snake venom disintegrin for integrin receptors. The crystal structure of trimestatin should provide a useful framework for designing and developing more effective drugs for controlling platelet aggregation and anti-angiogenesis cancer.

Divalent cations and the relationship between αA and βA domains in integrins

Integrins contain either one or two von Willebrand factor A-like domains, which are primary ligand and cation binding regions in the molecules. Here we examine the first structure of an A domain of a β subunit, in ανβ3 and compare it to known A domain structures of α subunits. Ligand binding to immobilized ανβ3 domain is stimulated by Ca2+ rather than inhibited by it. Biochemical, cell biological and structural evidence suggests that the A domain is a major site of ligand interaction in ανβ3. The Arg–Gly–Asp based inhibitor cilengitide (EMD 121974) inhibites ligand interaction with transmembrane-truncated ανβ3 in the presence of either Ca2+ or Mn2+ ions, and does so with similar kinetics. The ανβ3 structure reveals that both the αA and βA domains share common structural cores. But, in contrast to αA, the βA domain has three cation binding sites, that are involved either directly or indirectly in ligand binding. Structural alignment of αA and βA domains reveals additional loops unique only to the βA domain and much evidence support that that these loops are important for ligand binding specificity and for the interaction between α and β subunits. Since the position of these loops are evolutionary conserved but their primary sequence varies between the various βA domains, they represents potential targets for dissecting functional diversity among integrins.

Does the Integrin αA Domain Act as a Ligand for its βA Domain?

We thank Nancy Hogg for providing mAb 24 and Martyn K. Robinson for the KIM185 and 127 mAbs. This research was supported by grants from the National Institutes of Health (NIDDK, NIAID and NHLBI).