Structural basis of the differential binding of engineered knottins 2.5F and 2.5D to integrins αVβ3 and α5β1

Integrins αVβ3 and α5β1 play critical roles in tumor survival, invasion, metastasis, and angiogenesis and are validated targets for cancer therapy and molecular imaging. Increasing evidence suggests that targeting both integrins simultaneously with antagonists is more effective in cancer therapy because of concerns about resistance and paradoxical promotion of tumor growth with use of agents highly selective for a single integrin. Engineered Arg-Gly-Asp (RGD)-containing 3.5 kDa cysteine-knot proteins (knottins 2.5F and 2.5D) are attractive drug candidates due to their exceptional structural stability and high affinity binding to certain integrins. 2.5F binds both αVβ3 and α5β1, whereas 2.5D is αVβ3-selective. To elucidate the structural basis of integrin selection, we determined the structures of 2.5F and 2.5D both as apo-proteins and in complex with αVβ3. These data, combined with MD simulations and mutational studies, revealed a critical role of two αVβ3-specific residues in the vicinity of the metal ion dependent adhesion site (MIDAS) in promoting an αVβ3-induced fit of 2.5D. In contrast, conformational selection accounted for the specificity of 2.5F to both integrins. These data provide new insights into the structural basis of integrin-ligand binding specificity, and could help in development of integrin-targeted therapeutics.