Characterizing the binding of glycoprotein VI with nanobody 35 reveals a novel monomeric structure of glycoprotein VI where the conformation of D1+D2 is independent of dimerization

Damaskinaki, Foteini Nafsika; Jooss, Natalie J.; Martin, Eleyna M.; Clark, Joanne C.; Thomas, Mark R.; Poulter, Natalie S.; Emsley, Jonas; Kellam, Barrie; Watson, Steve P.; Slater, Alexandre

Abstract

Background: The platelet–signaling receptor glycoprotein VI (GPVI) is a promising antithrombotic target. We have previously raised a series of high-affinity nanobodies (Nbs) against GPVI and identified Nb2, Nb21, and Nb35 as potent GPVI inhibitors. The Nb2 binding site has been mapped to the D1 domain, which is directly adjacent to the CRP binding site. Ligand–binding complementary determining region 3 has only 15% conservation between all 3 Nbs. Objectives: To map the binding sites of Nb21 and Nb35 on GPVI. Methods: We determined the X-ray crystal structure of the D1 and D2 extracellular domains of the GPVI-Nb35 complex. We then looked at the effects of various GPVI mutations on the ability of Nbs to inhibit collagen binding and GPVI signaling using surface binding assays and transfected cell lines. Results: The crystal structure of GPVI bound to Nb35 was solved. GPVI was present as a monomer, and the D1+D2 conformation was comparable to that in the dimeric structure. Arg46, Tyr47, and Ala57 are common residues on GPVI targeted by both Nb2 and Nb35. Mutating Arg46 to an Ala abrogated the ability of Nb2, Nb21, and Nb35 to inhibit collagen–induced GPVI signaling and blocked the binding of all 3 Nbs. In addition, Arg60 was found to reduce Nb21 inhibition but not the inhibition Nb2 or Nb35. Conclusions: These findings reveal key residues involved in the high-affinity binding of GPVI inhibitors and negate the idea that GPVI dimerization induces a conformational change required for ligand binding.

• Journal Article Type: Article
• Acceptance Date: Nov 6, 2022
• Online Publication Date: Dec 22, 2022
• Publication Date: Feb 11, 2023
• Deposit Date: Jun 15, 2023
• Journal: Journal of Thrombosis and Haemostasis
• Print ISSN: 1538-7933
• Electronic ISSN: 1538-7836
• Peer Reviewed: Peer Reviewed
• Volume: 21
• Issue: 2
• Pages: 317-328
• DOI: https://doi.org/10.1016/j.jtha.2022.11.002
• Keywords: dimerization, glycoprotein VI, nanobody, thrombosis, platelet activation
• Public URL: https://nottingham-repository.worktribe.com/output/17372863
• Publisher URL: https://www.jthjournal.org/article/S1538-7836(22)07187-2/fulltext
• Related Public URLs: https://www.sciencedirect.com/science/article/abs/pii/S1538783622071872