Slater Alexandre, Di Ying, Clark Joanne C, Jooss Natalie J, Martin Eleyna M, Alenazy Fawaz, Thomas Mark R, Ariëns Robert A S, Herr Andrew B, Poulter Natalie S, Emsley Jonas, Watson Steve P
Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, United Kingdom.
Blood. 2021 Jun 17;137(24):3443-3453. doi: 10.1182/blood.2020009440.
Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C' loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.
糖蛋白VI（GPVI）是血小板上胶原蛋白的主要信号受体。我们针对重组GPVI-Fc（二聚体GPVI）制备了54个纳米抗体（Nb），根据其互补决定区3环将它们分为33个结构类别，并对它们结合重组GPVI、静息和活化血小板的能力以及抑制胶原蛋白诱导的血小板活化的能力进行了表征。来自6种不同结合类别的Nb对重组GPVI-Fc表现出最强的结合，这表明不存在单一的优势类别。最有效的3个Nb，即Nb2、21和35，以纳摩尔级的半数最大抑制浓度（IC50）值抑制胶原蛋白诱导的血小板聚集，并在流动条件下抑制血小板聚集。最有效的Nb，即Nb2，对重组单体和二聚体GPVI的结合解离常数（KD）分别为0.6和0.7 nM。单体GPVI与Nb2复合物的晶体结构揭示了一个与D1结构域内胶原蛋白相关肽（CRP）结合凹槽相邻的结合表位。此外，还观察到了一种涉及D2结构域之间结构域交换的GPVI新构象。C-C'环的向外延伸促进了结构域交换，C-C'环形成了结构域交换铰链。通过截短铰链区域使结构域交换无法发生，从而测试了这种构象的功能意义。Nb2仍然能够取代胶原蛋白和CRP与突变体的结合，但在基于细胞的NFAT报告基因检测中信号被消除。这表明C-C'环区域对GPVI信号传导很重要，但对配体结合不重要，并表明结构域交换结构可能代表一种活性GPVI构象。
