Conformational landscape of integrin α4β1 and its recognition of VCAM and conformation-stabilizing small molecules

Integrin α4β1 recognition of vascular cell adhesion molecule (VCAM) is essential for leukocyte adhesion, migration, and signaling, yet is structurally unresolved. Here, we report cryo-electron microscopy (cryo-EM) structures of human α4β1 in apo closed conformations and in an open conformation bound to conformation-specific antibody fragment 12G10, alone or together with VCAM domains 1-3 or 4-6. The homologous D1 and D4 domains recognize α4β1 with comparable affinity, inserting lengthwise between the α4 propeller and β1 βI domains. The VCAM loop that binds the integrin metal ion-dependent adhesion site (MIDAS) flips its backbone and Asp sidechain to coordinate the MIDAS Mg2+ ion. Crystal structures of α4β1 with small-molecule stabilizers of closed or open states reveal a novel closure-stabilizing motif and diverse binding modes, including to α/β interfaces outside the VCAM binding pocket. We define the mechanism of α4β1 activation and ligand recognition, providing a framework for the rational design of immunomodulatory therapeutics.