The affinity of S protein of new coronavirus with ACE2 is 10-20 times that of SARS

Novel coronavirus (2019-nCoV China) outbreak recently has been listed as an international public health emergency This virus can have a fever, severe respiratory disease and pneumonia symptoms Coronavirus S glycoprotein (spike glycoprotein) is a key target for developing new vaccines, therapeutic antibodies and diagnostic techniques On February 19, an article entitled "cryo EM structure of the 2019 ncov spike in the fusion" was published in science magazine The novel coronavirus S protein was analyzed by joint research and cryopem electron microscopy After in-depth study, researchers found that the affinity between the new crown virus S protein and host cell ACE2 was 10-20 times that of SARS, and the related research results were in the late stage, "Conformation", a research team from University of Texas and the National Institute of allergy and infectious diseases Vaccines and new antiviral drugs provide new ideas and research basis Structure of a novel coronavirus S protein in fusion conformation Photo source: Daniel wrap, et al According to WHO Novel coronavirus novel coronavirus (2019-nCoV) is considered to be highly coincident with a variety of bat coronavirus and SARS coronavirus in February 10th It is highly similar to other bat coronavirus and SARS coronavirus Compared with SARS coronavirus, the new coronavirus seems to be more easily transmitted by human beings, and it can be transmitted on several continents faster than the SARS coronavirus It will spread rapidly and let who declare a global public health emergency The novel coronavirus can be used to enter the host cell by using a tightly glycosylated homologous trimer I fusion protrusion protein (S) S protein can exist in a relatively stable pre conformation and undergo severe structural rearrangement to promote fusion of the membrane structure and host cell membrane, which is induced by the combination of S1 subunit and host receptor It can destroy the stability of the trimer before fusion, resulting in the loss of S1 subunit and the transformation of S2 subunit into a highly stable post fusion conformation In order to bind to the host receptor, the receptor binding domain of S1 undergoes a chain like conformational movement, which temporarily hides or exposes the determinants of receptor binding These two states are considered as "up" or "down" conformations, in which the down conformation corresponds to the inaccessible state of the receptor, while the up conformation corresponds to the reachable state of the receptor People are considered unstable Because of its indispensable function, S protein is expected to be a sensitive target of antibody mediated neutralization, and the S structure before fusion can provide atomic level information to help scientists design and develop vaccines Based on the novel coronavirus genome novel coronavirus reported by researchers, the researchers expressed the residues of the new extracellular domain 1-1208 of the coronavirus In addition, based on the previous stabilization strategy (which is very effective against the S protein of beta Coronavirus), two stable proline mutations were added to the C end of the S2 fusion machine, and the researchers obtained about 0 of the 293 cells At the same time, the proteins were purified by affinity chromatography and size exclusion chromatography Then the Cryo EM (frozen electron microscopy) grid was prepared by using purified and glycosylated s protein When the researchers collected and processed 3207 micrographs, they obtained a reconstruction image of a 3.5-angstrom-resolution 3D asymmetric trimer, in which a single receptor binding domain can be observed in the "up" domain Due to the small size of the receptor binding domain (0-21kda), the symmetry of this conformation may not be the same before 3D reconstruction and classification Obvious With the help of 3D variability features in cryosparc V2 algorithm, researchers can observe the structure of S1 subunit when the receptor binding domain moves in a chain, which may cause S1 to show a low partial resolution compared with the more stable S2 The novel coronavirus MERS coronavirus and even the far more porcine epidemic diarrhea virus can be observed in the seemingly random receptor binding domain The results observed in the new coronavirus S protein indicate that it may have the same induction mechanism believed to be conserved in the coronavirus family, where the binding of receptor to the exposed receptor binding domain will be SARS It leads to the production of unstable conformation, which leads to the shedding of S1 subunit and the refolding of S2 subunit The novel coronavirus seems to be a symmetrical trimer because of its S2 subunit, so the researchers have refined the 3D to give it C3 symmetry, thus generating a map of 3.2 AA resolution The S2 subunit has a good density By using the above map, researchers have constructed most of the extracellular domain of the new coronavirus, including each trimer The 66 novel coronavirus sites linked to glycosylation sites were 44 glycosylation sites The final model of the researchers crossed the S residue 22-1146 (site), which omitted some flexible loops The density of the S protein of the new coronavirus began to decrease after joining the extracellular domain structure of all the coronaviruses reported before, and thus reflected the seven peptide weight in the pre conformation The flexibility of HR2 (heptad repeat 2) The overall structure of novel coronavirus S protein is similar to the structure of SARS coronavirus S protein The rms deviation (RMSD) of both proteins on more than 959 C alpha atoms is 3.8 (A) The largest difference between them is the conformational difference between the receptor binding domain of their respective down conformation, and the SARS conjunct receptor binding junction in the "down" conformation The novel coronavirus novel coronavirus receptor binding domain is closer to the central cavity of homologous trimers Although there is a certain conformational difference, when the new single coronavirus S protein domain is aligned with the corresponding domain of the SARS coronavirus S protein, they can reflect the high level of the two proteins between the N proteins Degree of structural homology The novel coronavirus and the novel coronavirus RaTG13 S protein have a homology of about 96% The most obvious change is the insertion of S1/S2 protease cleavage site, which leads to the recognition of the "RRAR" pairs of basic amino acid proteases, rather than the single arginine sites of SARS coronavirus A similar phenomenon was observed, that is, amino acid insertion which can produce multiple pairs of alkaline amino acid protease sites was found in the relevant sites of highly toxic avian influenza virus and human influenza virus Novel coronavirus novel coronavirus S protein and RaTG13 S protein still exist in 29 residues, except 17 residues inserted in the S1/S2 junction The residues of 17 can be mapped to the receptor binding domain Subsequently, the researchers analyzed the sequences of 61 new coronavirus S proteins from the global shared avian influenza Initiative (GISAID) The novel coronavirus has only 9 amino acid substitutions, most of which are conservative Moreover, the researchers predict that it will not have a significant effect on the function or structure of the S protein of the new coronavirus Studies have shown that novel coronavirus S protein and SARS coronavirus S protein have the same function as host cell receptor, namely ACE2 (angiotensin converting enzyme2), which has prompted researchers to adopt surface plasmon resonance technology (SPR, surface plasmon), and ACE2 angiotensin converting enzyme2 The novel coronavirus S protein was constructed by resonance The results showed that the affinity of ACE2 and the extracellular domain structure of the new coronavirus S protein was 15 nM (equilibrium dissociation constant), which was 10-20 times the binding affinity between ACE2 and SARS coronavirus S protein extracellular domain structure The novel coronavirus novel coronavirus S protein extracellular domain complex was also synthesized and observed by negative electron microscopy The results showed that the structure of the -ACE2 complex of SARS coronavirus S protein was similar to that of the SARS coronavirus -ACE2 (high resolution cryopreservation) The high affinity of the new coronavirus to human ACE2 may help To explain why the virus is easy to spread among people, but further research is needed to clarify later The novel coronavirus S novel coronavirus S protein novel coronavirus S protein and the overall structural homology and shared receptor can help us detect the SARS SARS coronavirus receptor binding domain directed monoclonal antibody and new coronavirus receptor binding domain intersecting characteristics; at present, researchers have been on the new coronavirus receptor binding domain SD1 fragment ( S protein residue 319-591) was recombined and the binding level of ACE2 was determined by bli to confirm the folding of the recombinants Then bli was used to evaluate the cross reactivity of monoclonal antibodies S230, m396 and 80R targeting the receptor binding domain of SARS coronavirus Although novel coronavirus and novel coronavirus novel coronavirus have higher homology between the receptor binding domains, the binding of these three antibodies to the new coronavirus receptor binding domain was not found at the concentration of 1uM Although the three antibody epitopes only represent the smaller surface area of the new receptor coronavirus receptor binding domain, SARS has a higher degree of structural homology Novel coronavirus antibodies are not necessarily cross reactive, and researchers in the late stage may also use the S protein of new coronavirus as a probe to isolate and treat antibodies, SARS research shows The spread of novel coronavirus novel coronavirus has led WHO to identify itself as an international public health emergency At present, researchers urgently need to develop new vaccines and therapeutic methods for the virus At the atomic level, we elaborate the fine structure of the new coronavirus S protein or help to develop new antiviral therapy and vaccine in the later stage reference material: