A pivotal role for a conserved bulky residue at the 1-helix of the I integrin domain in ligand binding

The ligand-binding I and I domains of integrin are the best-studied von Willebrand factor A domains undergoing significant conformational changes for affinity regulation. In both I and I domains, the 1- and 7-helixes work in concert to shift the metal-ion-dependent adhesion site between the resting and active states. An absolutely conserved Gly in the middle of the 1-helix of I helps maintain the resting I conformation, whereas the homologous position in the I 1-helix contains a conserved Phe. A functional role of this Phe is structurally unpredictable. Using (L2) integrin as a model, we found that the residue volume at the Phe position in the 1-helix is critical for (L2) activation because trimming the Phe by small amino acid substitutions abolished (L2) binding with soluble and immobilized intercellular cell adhesion molecule 1. Similar results were obtained for (M2) integrin. Our experimental and molecular dynamics simulation data suggested that the bulky Phe acts as a pawl that stabilizes the downward ratchet-like movement of 6-7 loop and 7-helix, required for high-affinity ligand binding. This mechanism may apply to other von Willebrand factor A domains undergoing large conformational changes. We further demonstrated that the conformational cross-talk between (L) I and (2) I could be uncoupled because the (2) extension and headpiece opening could occur independently of the I activation. Reciprocally, the I activation does not inevitably lead to the conformational changes of the (2) subunit. Such loose linkage between the I and I is attributed to the I flexibility and could accommodate the (L2)-mediated rolling adhesion of leukocytes.