Abstract
Platelet specific integrin αIIbβ3 plays an essential role in hemostasis and thrombosis. It has been used as a prototype for understanding integrin activation and conformational regulation. Crystal structures of αIIbβ3 headpiece composed of the αIIb β-propeller and β3 βI, hybrid, and PSI domains in the absence or presence of RGD-mimetic drugs revealed the headpiece changing from a closed to an open conformation upon ligand binding. A striking change is the swing-out motion of the β3 hybrid domain away from the βI and the αIIb thigh domains. This is accompanied by the changing of the α1/α1'-helix from a bent to a merged and straightened α-helical conformation. The α1/α1'-helix is bent at the α1/α1' junction (β3 Ile-131 to Gly-135) as revealed by the crystal structures of β3, β1, β2, and β7 integrins in the closed headpiece conformation. The β3 Gly-135 at the α1/α1' junction is completely conserved among all the β integrins. We propose that the conserved glycine at the α1/α1' junction is critical for maintaining the bent α1/α1'-helix conformation, and the α1/α1'-helix unbending is required for integrin activation and bidirectional signaling. To test this hypothesis, we mutated the β3 Gly-135 to alanine and showed that the β3-G135A mutation rendered αIIbβ3 integrin constitutively binding the activation-dependent mAb PAC-1. In contrast, the β3-G135P mutation had minor effect on integrin activation. This is consistent with the idea that alanine tends to stabilize a straight α-helical structure, while glycine and proline tend to introduce a bend or kink into the α-helical conformation when present at the internal positions of an α-helix. That is, the conserved β3 Gly-135 is essential for restraining the α1/α1'-helix in the bent conformation. The β3 Gly-135 is partially exposed in the bent conformation of α1/α1'-helix and buried deeply into the hydrophobic environment upon the α1/α1'-helix unbending. We rationalized that the hydrophilic substitutions will restrain, while the hydrophobic substitutions will facilitate the burying of β3 Gly-135, and thus block and induce α1/α1'-helix unbending, respectively. As expected, the β3-G135R and G135K mutations completely blocked PAC-1 binding to αIIbβ3 integrin stimulated by Mn2+ or by the αIIb-R995D mutation that mimics integrin inside-out activation. In sharp contrast, the β3-G135L and G135M mutations constitutively induced PAC-1 binding to αIIbβ3 integrin. To further confirm the α1/α1'-helix unbending is required for integrin activation and signaling, we introduced tandem double or triple glycine substitutions into the α1/α1' junction to reinforce the bent conformation of α1/α1'-helix. Remarkably, all the double or triple glycine mutations completely abolished soluble PAC-1 binding stimulated by Mn2+ from outside or by the αIIb-R995D or αIIb-F993A mutation from inside the cell. This data provide compelling evidence that the integrin α1/α1'-helix unbending is indispensible for high affinity ligand binding. Interestingly, the β3-G135R or double glycine mutant still mediated cell adhesion to immobilized PAC-1 or fibrinogen, but at a reduced level. The cell adhesion could be blocked by eptifibatide, indicating the binding ability of the mutant integrins with the high affinity small molecule ligand. However, eptifibatide failed to induce the ectodomain extension of the mutant integrins. In addition, integrin-mediated outside-in signaling, such as cell spreading, focal adhesion and F-actin stress fiber formation, and focal adhesion kinase activation was inhibited by the β3-G135R or double glycine mutations. This data demonstrated that the conformational communication initiated by ligand binding is interrupted due to the defect of α1/α1'-helix unbending. We further showed that overexpression of talin1 head domain failed to induce PAC-1 binding to the αIIbβ3 integrin with double glycine mutations at the α1/α1' junction, but still induced integrin ectodomain extension. That is, in the inside-out integrin activation, the ectodomain extension alone does not result in high affinity ligand binding. The conformational signal has to be relayed to the ligand binding site through α1/α1'-helix unbending. In conclusion, our data established the structural role of the α1/α1' junction that allows relaxation of the α1/α1'-helix in the resting state and transmission of bidirectional conformational signals by helix unbending upon integrin activation.
Disclosures:
No relevant conflicts of interest to declare.
Multiple domains of the N-formyl peptide receptor are required for high-affinity ligand binding. Construction and analysis of chimeric N-formyl peptide receptors.
