9rn6 | pdb_00009rn6
Crystal structure of a protein mimic of SARS-CoV-2 spike's HR1 domain in complex with two nanobodies bound to different epitopes
Structural highlights
FunctionSPIKE_SARS2 attaches the virion to the cell membrane by interacting with host receptor, initiating the infection (By similarity). Binding to human ACE2 receptor and internalization of the virus into the endosomes of the host cell induces conformational changes in the Spike glycoprotein (PubMed:32142651, PubMed:32075877, PubMed:32155444). Uses also human TMPRSS2 for priming in human lung cells which is an essential step for viral entry (PubMed:32142651). Proteolysis by cathepsin CTSL may unmask the fusion peptide of S2 and activate membranes fusion within endosomes.[HAMAP-Rule:MF_04099][1] [2] [3] mediates fusion of the virion and cellular membranes by acting as a class I viral fusion protein. Under the current model, the protein has at least three conformational states: pre-fusion native state, pre-hairpin intermediate state, and post-fusion hairpin state. During viral and target cell membrane fusion, the coiled coil regions (heptad repeats) assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes.[HAMAP-Rule:MF_04099] Acts as a viral fusion peptide which is unmasked following S2 cleavage occurring upon virus endocytosis.[HAMAP-Rule:MF_04099] Publication Abstract from PubMedThe formation of a six-helix bundle between the conserved heptad-repeat regions 1 and 2 (HR1 and HR2) in SARS-CoV-2 Spike's S2 subunit is essential for membrane fusion and represents a promising therapeutic target. Previously, we reported recombinant proteins named CoVS-HR1, which mimic the HR1 region and block its interaction with HR2, inhibiting viral fusion. Moreover, they are recognized by plasma antibodies from COVID-19 convalescent patients. In this work, we generated camelid heavy-chain-only antibody fragments (VHHs), also named nanobodies (NBs), against a CoVS-HR1 variant mimicking the full HR1 region. A first generation of selected NBs bound HR1 with high affinity and competed with HR2. Notably, this set of NBs exclusively recognized the C-terminal half of HR1, and two of them showed mild neutralizing activity in cell infection assays. Using a truncated CoVS-HR1 variant (N2C), we selected a second generation of NBs targeting specifically the N-terminal half of HR1. However, these NBs did not demonstrate neutralizing activity, possibly due to their low binding affinities. Several NB epitopes were delineated by hydrogenâdeuterium exchange and mass spectrometry analysis, and the crystal structure of a ternary complex between an HR1-mimetic protein and two NBs was determined, confirming competition with HR2. Intriguingly, we found cooperative binding effects between NBs targeting each half of HR1, but these did not result in detectable inhibitory synergy. These findings demonstrate the existence of neutralizing epitopes in the S2 HR1 region and provide a foundation for future development of enhanced neutralizing NBs focused on specific epitopes using HR1-mimetic proteins. Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit.,Polo-Megias D, Cano-Munoz M, Trolese P, Lestani S, La Rocchia I, Pierangelini A, Fongaro B, de Laureto PP, Morales-Yanez FJ, Vaneyck J, Vanderplasschen A, Decoville T, Laumond G, Salinas-Garcia MC, Camara-Artigas A, Gavira JA, Moog C, Dumoulin M, Conejero-Lara F Int J Biol Macromol. 2026 Jan;340(Pt 1):150022. doi: , 10.1016/j.ijbiomac.2025.150022. Epub 2026 Jan 6. PMID:41506506[4] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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