Sunday 17th of January 2021

covid-19 — science...

spikes   The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein enables viral entry into host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor and is a major target for neutralizing antibodies. About 20 to 40 spikes decorate the surface of virions.


Turoňová et al. now show that the spike is flexibly connected to the viral surface by three hinges that are well protected by glycosylation sites. The flexibility imparted by these hinges may explain how multiple spikes act in concert to engage onto the flat surface of a host cell.



The spike surface protein (S) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required to initiate infection (1). It binds to the angiotensin-converting enzyme 2 (ACE2) (23) to mediate viral entry. S also determines tissue and cell tropism. Mutations may alter the host range of the virus and enable the virus to cross species barriers (45). Vaccine efforts focus on neutralizing antibodies that block infection by binding to S.

S is a trimeric class I viral fusion protein (6) with a club-like shape of ~20 nm in length. The ectodomain consists of a head, which has been extensively studied in vitro. It is connected to the membrane by a slender stalk. The three receptor binding domains (RBDs) of the S head are conformationally variable, which may relate to receptor binding. In the closed conformation, the RBDs are shielded by the N-terminal domains (NTDs). In the open conformation, one RBD is exposed upward away from the viral membrane (23). Previous studies resolved roughly two-thirds of the predicted 22 N-linked glycans that are thought to shield S against antibodies (23). It remains unknown whether the distribution of the conformational states and the glycosylation pattern observed with recombinant protein in vitro are representative of the native state generated during viral assembly. Furthermore, little is known about the stalk of S and how its conformational variability within the virion may affect the accessibility of epitopes for neutralizing antibodies and facilitate viral entry.

SARS-CoV-2 virions present prefusion S in an irregular pattern

To structurally analyze SARS-CoV-2 S in situ, we passaged the virus through tissue culture cells and used sucrose centrifugation to purify it from the inactivated supernatant (see materials and methods). We acquired a large-scale cryo–electron tomography dataset that consists of 266 tilt series covering >1000 viruses. Visual inspection of the tomographic reconstructions revealed a very-high-quality data set in which individual protein domains were clearly visible (Fig. 1A and movie S1). On average, 40 copies of the S trimer resided on the surface. S proteins appeared to be distributed randomly on the viral surface without any significant tendency to cluster (Fig. 2A).

S was mostly present in the prefusion conformation (Fig. 1B). Postfusion conformations (78) were very rare (<0.1%), which appears typical for Vero E6 host cells (9). Sanger sequencing and immunoblot analysis revealed that the furin site for proteolytic cleavage into the S1 and S2 fragments (510) was lost during tissue culture passage (Fig. 1C and fig. S1), consistent with previous studies (1112). However, the isolate contained the Asp614→Gly (D614G) allele (1314).Large-scale sequencing of RNA isolated from tissue culture supernatant confirmed both findings (supplementary materials).

Subtomogram averaging with NovaSTA (15) and STOPGAP (16) resulted in a cryo–electron microscopy (cryo-EM) map of the S head at 7.9 Å resolution (fig. S2), in which secondary structure elements and individual glycosylation sites were clearly discernible (Fig. 2, B and C). Classification suggested that about half of S was present in the fully closed conformation. A considerable fraction of the remaining subtomograms had one RBD exposed (fig. S3). Structural analysis of the asymmetric unit yielded an average map of the closed conformation at an overall resolution of 4.9 Å. In particular, the cluster of parallel helices in the center of the head was clearly resolved (Fig. 2Dand fig. S4).

By contrast, the stalk connecting the S head to the viral membrane appeared to be dynamic. Although the head was fully contained in the tomographic map, only the top of the stalk domain was resolved. Emerging from the neck of the spike head, it contains an 11-residue Leu repeat sequence (L1141, L1145, and L1152) and adopts an unusual right-handed coiled coil, consistent with a recent single-particle structure of the S head (7). We will henceforth refer to this part of the stalk domain as the “upper leg.” Right-handed trimeric coiled coils were long thought to be absent from the structural proteome (17) but can be seen in the postfusion structure of S from the related mouse hepatitis virus (18).

A stalk with three flexible hinges connects S to the viral membrane

The tomographic images suggest the presence of flexible hinges in the stalk. Stalks of individual S proteins are clearly visible in the tomograms (Fig. 1B), but, after averaging, their density declined sharply at the end of the trimeric coiled coil that forms the upper leg (Fig. 2B). Moreover, the head exhibited large positional and orientational freedom. It was tilted up to ~90° with respect to the normal at distances of 5 to 35 nm from the membrane (Fig. 2E). We grouped our subtomograms into four classes, according to their distance from the bilayer, and averaged them separately. At an intermediate distance, parts of the stalk and bilayer were resolved, suggesting a more defined conformation (Fig. 2F). We then subselected ~3200 particles in which the head was oriented roughly perpendicular to the membrane. In the resulting average, the stalk domain was resolved (fig. S5A). Visual inspection of the respective subtomograms, in which the stalk domains are clearly observed, further corroborated the idea of a kinked stalk with potentially several hinges (Fig. 2F). Local refinement of the lower part of the stalk (henceforth referred to as the “lower leg”) resulted in a moderately resolved structure that would be consistent with the continuation of the coiled coil below a flexible hinge (henceforth referred to as the “knee”) (fig. S5B).

Molecular dynamics (MD) simulations helped us to pinpoint the molecular origins of the flexibility seen in the tomograms. We performed a 2.5-μs-long all-atom MD simulation of a 4.1 million atom system containing four glycosylated S proteins anchored into a patch of viral membrane and embedded in aqueous solvent (Fig. 3A). In the simulations, the S heads remained stable. The stalks, however, exhibited pronounced hinging motions at the junctions between the S head and the upper leg (“hip”), between the upper and lower legs (“knee”), and between the lower leg and the transmembrane domain (“ankle”). This observation was consistent with discrete leg segments seen in the raw tomograms (Fig. 3, B and C). The hip joint flexed the least (16.5° ± 8.8°), followed by the ankle (23.0° ± 11.7°) and the knee (28.4° ± 10.2°) (Fig. 3D and fig. S6). However, the limited sampling in the MD simulation may not have covered the full range of motions (compare Fig. 2E and fig. S6D).



Read and see more:



This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the spikes...



Read and see more:



This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.