EM Structure of the Ectodomain of Integrin CD11b/CD18 and Localization of Its Ligand-Binding Site Relative to the Plasma Membrane

One-half of the integrin alpha-subunit Propeller domains contain and extra vWFA domain (alpha A domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS). We used electron microscopy to determine the 3D structure of the alpha A-containing ectodomain of the leukocyte integrin CD11b/CD18 (alpha M beta 2) in its inactive state. A well defined density for alpha A was observed within a bent ectodomain conformation, while the structure of the ectodomain in complex with the Fab fragment of mAb107, which binds at the MIDAS face of CD11b and stabilizes the inactive state, further revealed that alpha A is restricted to a relatively small range of orientations relative to the Propeller domain. Using Fab 107 as probe in fluorescent lifetime imaging microscopy (FLIM) revealed that alpha A is positioned relatively far from the membrane surface in the inactive state, and a systematic orientation search revealed that the MIDAS face would be accessible to extracellular ligand in the inactive state of the full-length cellular integrin. These studies are the first to define the 3D EM structure of an alpha A-containing integrin ectodomain and to position the ligand-binding face of alpha A domain in relation to the plasma membrane, providing new insights into current models of integrin activation.