Abstract
One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA 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 αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMβ2) in its inactive state. A well defined density for αA 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 αA 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 αA 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 αA-containing integrin ectodomain and to position the ligand-binding face of αA domain in relation to the plasma membrane, providing new insights into current models of integrin activation.
Highlights
Integrins are non-covalent ab heterodimeric cell adhesion receptors that regulate diverse biological processes by signaling bidirectionally across the plasma membrane
The Propeller domain is followed by a Thigh- and Calf-1 and Calf-2 domains, a transmembrane (TM) segment ending with a short cytoplasmic tail. bA is inserted in an Ig-like Hybrid domain, which is flanked by an N-terminal PSI domain and followed by four EGF-like domains (EGF1-4), a beta-tail domain, a TM segment and terminating with a short cytoplasmic tail
In this report, we have used electron microscopy and 3D image reconstruction to determine the 3D structure of inactive CD11b/ CD18 ectodomain and position its ligand-binding CD11bA domain within the integrin head
Summary
Integrins are non-covalent ab heterodimeric cell adhesion receptors that regulate diverse biological processes by signaling bidirectionally across the plasma membrane (reviewed in [1]). Crystal structure of aVb3 [2] and aIIbb ectodomains [3] revealed that integrins adopt a compact bent conformation that consists of a ligand-binding ‘head’ comprising a seven-bladed Propeller domain from the a-subunit bound noncovalently to a vWFA domain (bA domain) from the b-subunit. The Propeller domain is followed by a Thigh- and Calf-1 and Calf-2 domains, a transmembrane (TM) segment ending with a short cytoplasmic tail. Mg2+dependent binding of physiologic ligands takes place at the integrin head, and requires a metal-ion-dependent-adhesion-site (MIDAS) in the bA domain [5]. In nine of the twenty-four mammalian integrins, an additional vWFA domain (aA domain) is inserted in the Propeller domain, and acts through its MIDAS face as the principal ligand-binding site [6]
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