Electron microscopy shows that binding of monoclonal antibody PT25-2 primes integrin αIIbβ3 for ligand binding
| Autor: | Tetsuji Kamata, Makoto Handa, Jihong Li, Thomas Walz, Dragana Nesic, Marta Filizola, Aleksandar Spasic, Barry S. Coller, Martin Bush |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
medicine.drug_class Integrin Platelet Glycoprotein GPIIb-IIIa Complex 030204 cardiovascular system & hematology Ligands Monoclonal antibody Epitope Mice 03 medical and health sciences 0302 clinical medicine medicine Animals Binding site Receptor biology Chemistry Cryoelectron Microscopy Mutagenesis Antibodies Monoclonal Hematology Platelets and Thrombopoiesis Ligand (biochemistry) Kinetics A-site 030104 developmental biology biology.protein Biophysics |
| Zdroj: | Blood Adv |
| ISSN: | 2473-9537 2473-9529 |
| DOI: | 10.1182/bloodadvances.2020004166 |
| Popis: | The murine monoclonal antibody (mAb) PT25-2 induces αIIbβ3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that PT25-2 binds to the αIIb β propeller, a site distant from the Arg-Gly-Asp–binding pocket. To elucidate the mechanism, we studied the αIIbβ3–PT25-2 Fab complex by negative-stain and cryo-electron microscopy (EM). We found that PT25-2 binding results in αIIbβ3 partially exposing multiple ligand-induced binding site epitopes and adopting extended conformations without swing-out of the β3 hybrid domain. The cryo-EM structure showed PT25-2 binding to the αIIb residues identified by mutagenesis but also to 2 additional regions. Overlay of the cryo-EM structure with the bent αIIbβ3 crystal structure showed that binding of PT25-2 creates clashes with the αIIb 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 αIIbβ3 ligand binding by binding to extended conformations and by preventing the interactions between the αIIb and β3 leg domains and subsequently the βI and β3 leg domains required for the bent-closed conformation. |
| Databáze: | OpenAIRE |
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