Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, The Midlands, UK; Biodiscovery Institute, University Park, University of Nottingham, Nottingham, UK.
Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
J Thromb Haemost. 2023 Feb;21(2):317-328. doi: 10.1016/j.jtha.2022.11.002. Epub 2022 Dec 22.
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.
To map the binding sites of Nb21 and Nb35 on GPVI.
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.
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.
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.
血小板信号受体糖蛋白 VI（GPVI）是一种很有前途的抗血栓靶点。我们之前已经针对 GPVI 产生了一系列高亲和力纳米抗体（Nbs），并鉴定出 Nb2、Nb21 和 Nb35 是有效的 GPVI 抑制剂。Nb2 的结合位点已被定位到 D1 结构域，该结构域直接与 CRP 结合位点相邻。配体结合互补决定区 3 在所有 3 个 Nb 之间只有 15%的保守性。
确定 Nb21 和 Nb35 在 GPVI 上的结合位点。
我们测定了 GPVI-Nb35 复合物的 D1 和 D2 细胞外结构域的 X 射线晶体结构。然后，我们使用表面结合测定和转染细胞系，观察各种 GPVI 突变对 Nb 抑制胶原蛋白结合和 GPVI 信号的能力的影响。
我们解析了与 Nb35 结合的 GPVI 的晶体结构。GPVI 呈单体形式，D1+D2 构象与二聚体结构相似。Arg46、Tyr47 和 Ala57 是 Nb2 和 Nb35 靶向的 GPVI 的常见残基。Arg46 突变为 Ala 会破坏 Nb2、Nb21 和 Nb35 抑制胶原蛋白诱导的 GPVI 信号的能力，并阻断所有 3 个 Nb 的结合。此外，发现 Arg60 减少了 Nb21 的抑制作用，但不影响 Nb2 或 Nb35 的抑制作用。
这些发现揭示了涉及 GPVI 抑制剂高亲和力结合的关键残基，并否定了 GPVI 二聚化诱导配体结合所需构象变化的观点。
