Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVβ3 bound to a physiologic ligand: the 10th type III RGD-domain of wild-type fibronectin (wtFN10), or to a high affinity mutant (hFN10) that acts as a pure antagonist. Comparison of these structures revealed a central π - π interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the β3-subunit that blocked conformational changes triggered by wtFN10, and trapped hFN10-bound αVβ3 in an inactive conformation. Removing the Trp1496 or Tyr122 side-chains, or reorienting Trp1496 away from Tyr122, converted hFN10 into a partial agonist. The findings offer new insights on the mechanism of integrin activation and a basis for design of RGD-based pure antagonists.

Background: 17E6, a primate-specific mouse mAb that inhibits ␣V integrins, is in phase II trials for treating cancer. Results: We determined crystal structure of the ␣V␤3-17E6 Fab complex, revealing the molecular basis of 17E6 specificity and function. Conclusion: 17E6 is an allosteric inhibitor of fibronectin-integrin interaction. Significance: The defined 17E6 epitope may help in developing novel therapeutics targeting related regions in other integrins.

Supplemental Figures (#3) Figure S1. Crystal structures of αVβ3 bound to 2.5D or 2.5F. Composite simulated annealing omit maps (in grey isomesh) at 1.0 σ of 2.5D (in brown, A) and 2.5F (in (purple, B) in the knottin/αVβ3 structures (in ribbons) with only portions of the integrin propeller (blue) and βA (in green) domains shown. The MIDAS Mn 2+ ion is in cyan. Related to Figure 3.