7la4

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The murine monoclonal antibody (mAb) PT25-2 induces alphaIIbbeta3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that PT25-2 binds to the alphaIIb beta propeller, a site distant from the Arg-Gly-Asp-binding pocket. To elucidate the mechanism, we studied the alphaIIbbeta3-PT25-2 Fab complex by negative-stain and cryo-electron microscopy (EM). We found that PT25-2 binding results in alphaIIbbeta3 partially exposing multiple ligand-induced binding site epitopes and adopting extended conformations without swing-out of the beta3 hybrid domain. The cryo-EM structure showed PT25-2 binding to the alphaIIb residues identified by mutagenesis but also to 2 additional regions. Overlay of the cryo-EM structure with the bent alphaIIbbeta3 crystal structure showed that binding of PT25-2 creates clashes with the alphaIIb calf-1/calf-2 domains, suggesting that PT25-2 selectively binds to partially or fully extended receptor conformations and prevents a return to its bent conformation. Kinetic studies of the binding of PT25-2 compared with mAbs 10E5 and 7E3 support this hypothesis. We conclude that PT25-2 induces alphaIIbbeta3 ligand binding by binding to extended conformations and by preventing the interactions between the alphaIIb and beta3 leg domains and subsequently the betaI and beta3 leg domains required for the bent-closed conformation.
The murine monoclonal antibody (mAb) PT25-2 induces alphaIIbbeta3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that PT25-2 binds to the alphaIIb beta propeller, a site distant from the Arg-Gly-Asp-binding pocket. To elucidate the mechanism, we studied the alphaIIbbeta3-PT25-2 Fab complex by negative-stain and cryo-electron microscopy (EM). We found that PT25-2 binding results in alphaIIbbeta3 partially exposing multiple ligand-induced binding site epitopes and adopting extended conformations without swing-out of the beta3 hybrid domain. The cryo-EM structure showed PT25-2 binding to the alphaIIb residues identified by mutagenesis but also to 2 additional regions. Overlay of the cryo-EM structure with the bent alphaIIbbeta3 crystal structure showed that binding of PT25-2 creates clashes with the alphaIIb calf-1/calf-2 domains, suggesting that PT25-2 selectively binds to partially or fully extended receptor conformations and prevents a return to its bent conformation. Kinetic studies of the binding of PT25-2 compared with mAbs 10E5 and 7E3 support this hypothesis. We conclude that PT25-2 induces alphaIIbbeta3 ligand binding by binding to extended conformations and by preventing the interactions between the alphaIIb and beta3 leg domains and subsequently the betaI and beta3 leg domains required for the bent-closed conformation.
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Electron microscopy shows that binding of monoclonal antibody PT25-2 primes integrin alphaIIbbeta3 for ligand binding.,Nesic D, Bush M, Spasic A, Li J, Kamata T, Handa M, Filizola M, Walz T, Coller BS Blood Adv. 2021 Apr 13;5(7):1781-1790. doi: 10.1182/bloodadvances.2020004166. PMID:33760023<ref>PMID:33760023</ref>
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, PMID:33760023<ref>PMID:33760023</ref>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>

Current revision

Integrin AlphaIIbBeta3-PT25-2 Complex

PDB ID 7la4

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