Mapping the ligand-binding pocket of integrin α5β1 using a gain-of-function approach

Integrin alpha 5 beta 1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human integrin alpha 5 beta 1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by integrin alpha 5 beta 1 is poorly understood. In the present study, we demonstrate that zebrafish alpha 5 beta 1 integrins do not interact with human fibronectin or the human alpha 5 beta 1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish alpha 5 beta 1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish alpha 5 subunit beta-propeller domain shows that these residues are important for the recognition of the Arg-Gly-Asp (RGD) motif and the synergy sequence [Pro-His-Ser-Arg-Asn (PHSRN)] in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish integrin alpha 5 subunit with the corresponding regions of human alpha 5 we show that blades 1-4 of the beta-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the beta-propeller domain. We find that the loop connecting blades 2 and 3 of the beta-propller, the D3-A3 loop, contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human integrin alpha 5 beta 1 supports an important function for D3-A3 loop residues Trp(157) and Ala(158) in the binding of antagonists. These results will aid the development of reagents that block integrin alpha 5 beta 1 functions in vivo.