Person:

Zhu, Jianghai

The lymphocyte homing receptor integrin (\alpha_4 \beta_7) is unusual for its ability to mediate both rolling and firm adhesion. (\alpha_4 \beta_1) and (\alpha_4 \beta_7) are targeted by therapeutics approved for multiple sclerosis and Crohn’s disease. Here, we show by electron microscopy and crystallography how two therapeutic Fabs, a small molecule (RO0505376), and mucosal adhesion molecule-1 (MAdCAM-1) bind α4β7. A long binding groove at the (\alpha_4 -\beta_7)interface for immunoglobulin superfamily domains differs in shape from integrin pockets that bind Arg-Gly-Asp motifs. RO0505376 mimics an Ile/Leu-Asp motif in (\alpha_4) ligands, and orients differently from Arg-Gly-Asp mimics. A novel auxiliary residue at the metal ion–dependent adhesion site in (\alpha_4 \beta_7) is essential for binding to MAdCAM-1 in (Mg^{2+}) yet swings away when RO0505376 binds. A novel intermediate conformation of the (\alpha_4 \beta_7) headpiece binds MAdCAM-1 and supports rolling adhesion. Lack of induction of the open headpiece conformation by ligand binding enables rolling adhesion to persist until integrin activation is signaled.

Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin αIIbβ3 on platelets, resulting in platelet aggregation. αvβ3 binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's α subunit. αIIbβ3 also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the γ subunit (γC peptide). These distinct modes of fibrinogen binding enable αIIbβ3 and αvβ3 to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin αIIbβ3–γC peptide interface, and, for comparison, integrin αIIbβ3 bound to a lamprey γC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in γC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion–dependent adhesion site (MIDAS) Mg2+ ion binds the γC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca2+ ion binds the γC C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered γC peptide enhances our understanding of the involvement of γC peptide and integrin αIIbβ3 in hemostasis and thrombosis.

Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the αIIbβ3 integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca2+, α1 helix, α1’ helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward αIIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening.