Structural characterisation of a novel GPVI nanobody-complex reveals a biologically active domain-swapped GPVI dimer
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Colleges, School and Institutes
GPVI is the major signalling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 (CDR3) loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterised their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nanobodies from six different binding classes showed the strongest binding to recombinant GPVI-Fc suggesting that there was not a single dominant class. The most potent three, Nb2, 21 and 35, inhibited collagen-induced platelet aggregation with nanomolar 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 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 signalling was abolished in a cell-based NFAT-reporter assay. This demonstrates that the C-C’ loop region is important for GPVI signalling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.
Acknowledgements This research was funded, in whole or in part, by a Wellcome Trust Investigator Award (204951/B/16/Z). A CC BY licence is applied to the AAM arising from this submission, in accordance with the grant’s open access conditions. SPW is a British Heart Foundation Professor (CH 03/003). We acknowledge the Diamond Light Source for provision of synchrotron radiation in using the beamline I04. NJ is supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 766118, and is registered in the PhD programs of Maastricht and University of Birmingham.
|Publication status||Published - 27 Jan 2021|