Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1

Targeting both integrins alpha V beta 3 and alpha 5 beta 1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.5F binds selectively to alpha V beta 3 and alpha 5 beta 1, whereas knottin 2.5D is alpha V beta 3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with alpha V beta 3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages alpha V beta 3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.