Autonomous conformational regulation of beta3integrin and the conformation-dependent property of HPA-1a alloantibodies.

Integrin α/β heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale conformational rearrangement. During the inside-out activation, signals impinging on the cytoplasmic tail of β subunit induce the α/β separation at the transmembrane and cytoplasmic domains, leading to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrangement in the inside-out activation. The β 3 integrins, including α IIb β 3 and α V β 3 , are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the β 3 PSI domain defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically important bleeding disorders. Some, but not all, anti–HPA-1a alloantibodies can distinguish the α IIb β 3 from α V β 3 and affect their functions with unknown mechanisms. Here we designed a single-chain β 3 subunit that mimics a separation of α/β heterodimer on inside-out activation. Our crystallographic and functional studies show that the single-chain β 3 integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the β 3 subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain β 3 integrin, we identified the conformation-dependent property of anti–HPA-1a alloantibodies, which enables them to differently recognize the β 3 in the bent state vs. the extended state and in the complex with α IIb vs. α V . This study provides deeper understandings of integrin conformational activation on the cell surface.