Locking the β3 Integrin I-like Domain into High and Low Affinity Conformations with Disulfides

Abstract

Although integrin α subunit I domains exist in multiple conformations, it is controversial whether integrin β subunit I-like domains undergo structurally analogous movements of the α7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the β3 integrin I-like domain to lock its β6-α7 loop and α7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor αIIbβ3T329C/A347C was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded αIIbβ3V332C/M335C was locked in a high affinity state. The results suggest that activation of the β subunit I-like domain is analogous to that of the α subunit I domain, i.e. that axial movement in the C-terminal direction of the α7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.