In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges

Abstract

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. Here, we combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed pre-fusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines.

SARS-CoV-2 Simulations Go Exascale to Capture Spike Opening and Reveal Cryptic Pockets Across the Proteome

“…Capturing opening of S is an impressive technical feat given that previous large-scale simulations were unable to observe this essential event for the initiation of infection. 28,31,32 We successfully captured this rare event for both glycosylated and unglycosylated S and found that glycosylation slightly increases the population of the open state, but is qualitatively very similar to the unglycosylated ensemble (Fig. S1).…”

The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens

“…Together with the finding that although the SARS-CoV-2 S protein shares a high degree of amino acid sequence identity with that of SARS-CoV (~76% overall), the RBM is less conserved (~47% identity) than any other functional region or domain (82), it can thus been surmised that the RBM has the most immunodominant neutralizing epitope(s) of the whole S protein, capable of readily eliciting strong neutralizing antibody responses. However, the native trimeric SARS-CoV-2 S protein could conceal each of its immunodominant RBMs by adopting the closed conformation (41,83). Therefore, SARS-CoV-2 evades immune surveillance also through conformational masking, which is well-documented for HIV-1 (43,44); while at the same time, the S protein could transiently sample the functional state to engage ACE2, consistent with the notion that the fusion glycoprotein of highly pathogenic viruses have evolved to perform its functions while evading host neutralizing antibody responses.…”

Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures

“…Peplomer density of SARS-CoV-2 was determined recently by cryo-EM for different virus isolates than the virus isolate used in our study. The reported mean values vary from 25 to 40 peplomers per virus parNcle with significant variaNon among parNcles [11][12][13][14] . Our measurements of 9 peplomers per virus segment, represenNng roughly a third of the enNre virus, falls in the same range.…”

“…Biosafety sNll requires inacNvaNon of the virus preparaNon before conducNng the sample preparaNon for cryo-EM, and the effects on the ultrastructure must be carefully controlled. The recently published work on isolated SARS-CoV-2 [11][12][13] only parNally addressed those aspects 11 .…”

“…Very recently, morphometric data on isolated SARS-CoV-2 parNcles [10][11][12][13] and virus parNcles in cells 14 , obtained by cryo-EM, were published or became available as a preprint. While cryo-EM is definitely the best method to study virus ultrastructure and structural biology, convenNonal EM, using plasNc embedding, sNll is of relevance, especially for the study of samples, which cannot be easily analyzed by cryo-EM, such as complex mulNcellular objects or pathological material obtained from paNents.…”