Sterically confined rearrangements of SARS-CoV-2 Spike protein control cell invasion

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and transmission involves a series of processes that may be targeted by vaccines and therapeutics. During transmission, host cell invasion is controlled by a large-scale (200-300 Å) conformational change of the Spike protein. This conformational rearrangement leads to membrane fusion, which creates transmembrane pores through which the viral genome is passed to the host. During Spike-protein-mediated fusion, the fusion peptides must be released from the core of the protein and associate with the host membrane. While infection relies on this transition between the prefusion and postfusion conformations, there has yet to be a biophysical characterization reported for this rearrangement. That is, structures are available for the endpoints, though the intermediate conformational processes have not been described. Interestingly, the Spike protein possesses many post-translational modifications, in the form of branched glycans that flank the surface of the assembly. With the current lack of data on the pre-to-post transition, the precise role of glycans during cell invasion has also remained unclear. To provide an initial mechanistic description of the pre-to-post rearrangement, an all-atom model with simplified energetics was used to perform thousands of simulations in which the protein transitions between the prefusion and postfusion conformations. These simulations indicate that the steric composition of the glycans can induce a pause during the Spike protein conformational change. We additionally show that this glycan-induced delay provides a critical opportunity for the fusion peptides to capture the host cell. In contrast, in the absence of glycans, the viral particle would likely fail to enter the host. This analysis reveals how the glycosylation state can regulate infectivity, while providing a much-needed structural framework for studying the dynamics of this pervasive pathogen.

Keywords: COVID-19; energy landscapes; membrane fusion; molecular biophysics; structural biology; virus.

Figure 1.. Spike-protein-mediated membrane fusion.

(A) The active Spike protein assembly is composed of the subunits S1 (white surface) and S2 (cartoon representation) (Walls et al., 2020), which remain bound through nonbonded interactions. Numerous glycosylation sites (glycans shown in orange) are present in the Head Group (HG) and Heptad Repeat 2 (HR2) regions. The modeled glycans are consistent with previous studies (Casalino et al., 2020; Turoňová et al., 2020). (B) Upon recognition of the ACE2 receptor and cleavage at the S2’ site, S1 dissociates. In addition to the HG and HR2 regions, S2 is composed of the Heptad Repeat 1 (HR1), Linker (L), Fusion Peptide (FP), Connector (CR), and Transmembrane (TM) regions. Since the HR2 and TM regions were not resolved in the prefusion structure (PDB ID: 6VXX [Walls et al., 2020]), they were modeled as a helical bundle, consistent with previous studies (Casalino et al., 2020). (C) Release of S1 allows for the FPs to associate with and recruit the host membrane. The HG and HR1 regions undergo a large-scale rotation (>90 degrees), which leads to fusion of the host and viral membranes. Since the CR and FP regions were not resolved in the postfusion structure (PDB ID: 6M3W [Fan et al., 2020]), they were modeled as an extended helical bundle.

Figure 2.. Simulating Spike-protein-mediated membrane fusion. Simulations with an all-atom structure-based model (Whitford et al., 2009; Noel et al., 2016) allow for transitions between prefusion and postfusion configurations to be observed.

(A) Schematic representation of the energetics in the structure-based model. The postfusion configuration was defined as the global potential energy minimum. The pre-cleavage state (green) is assumed to be stable, where cleavage and release of S1 leads to an unstable prefusion configuration (black). While, in the employed model, stabilizing energetic terms favor the postfusion configuration, steric interactions between the protein and glycans may impede the motion (black vs. red). (B) Representative simulation (1 of 1000) of the pre-to-post transition. Spatial rmsd from the post configuration (excluding TM, CR, and FPs) is shown, as a function of time. The simulation included explicit glycans, as well as an effective viral membrane potential. (C) After an initial relaxation phase (panel B), the head (red) appears to become caged by the HR2 strands (gray), allowing it to sample configurations near the viral membrane (pink). While the host membrane (yellow) was not included in the simulations, it is depicted for illustrative purposes. (D) After reaching the caged ensemble, the head escapes and the HR1-HR2 superhelix assembles. The position of the head group, relative to the TM region (blue), is described by the cylindrical coordinates r_head and z_head. The origin is defined as the geometric center of TM, and the cylindrical axis is perpendicular to the viral membrane. See Materials and methods for details. (E) Probability distribution of simulated events (with glycans) reveals an obligatory cage-like intermediate.

Figure 3.. Glycan-induced caging of the head domain. Glycans impede head rearrangement by introducing a steric cage.

(A) Snapshot from the caged ensemble illustrates the high density of glycans surrounding the head. (B) To define the duration of each caging event (${\tau }_{\mathrm{cage}}={\tau }_{\mathrm{exit}}-{\tau }_{\mathrm{enter}}$), we measured z_head and r_head (Figure 2D). Based on the 2D probability distribution (Figure 2E), the system was defined as entering the cage when z_head first drops below 6.5 nm: ${\displaystyle {\tau }_{\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{r}}}$. ${\displaystyle {\tau }_{\mathrm{e}\mathrm{x}\mathrm{i}\mathrm{t}}}$ is the time at which the head moves laterally, relative to the trans-membrane region ($r_{\mathrm{head}}>5$ nm). (C) Distribution of ${\tau }_{\mathrm{cage}}$ values when glycans are present. There is an extended tail at large values ($100-500\mu s$). (D) When glycans are absent, the ${\tau }_{\mathrm{cage}}$ values are narrowly distributed around short timescales.

Figure 4.. Caging of head allows for extension of HR1 helix.

(A) Distribution of $Q_{\mathrm{HR1}}$ (number of postfusion-specific HR1 contacts) values when glycans are present. Distribution describes the first frame in each simulation for which the head is outside of the steric cage. In all but three simulations, nearly all HR1 contacts (>1420 of 1489) are formed upon exit of the cage. (B) Distribution when glycans are not included. When glycans are absent, it is common that HR1 is not completely formed (i.e. $Q_{\mathrm{HR1}}<$ 1350) prior to HG escape. (C) Representative snapshot of a caged structure in which HR1 is fully formed and extended toward the host. (D) Representative snapshot of a glycan-free case where the head escapes prior to fully forming HR1. As a result, HR1 can adopt bent configurations.

Figure 5.. Glycans promote host capture.

(A) Snapshot of the glycosylated Spike protein with the head domain in a caged configuration (glycans not shown). Caging allows the fusion peptide tails to extend toward and engage the host membrane. $d_{\mathrm{FP}}$ is the distance of the center of mass of each fusion peptide from the viral membrane surface. To calculate the probability of host capture, different values of the virion-host distance (d_host) were considered. (B) Representative simulated trajectory, showing $d_{\mathrm{FP}}$ for each of the fusion peptides in a single S2 subunit. For reference, a d_host value of 30 nm is indicated by a dashed line. (C) The probability of membrane fusion is expected to be proportional to the probability that at least one tail extends to the host membrane ($d_{\mathrm{FP}}>d_{\mathrm{host}}$). There is a higher probability of extending to larger d_host values when glycans are present (black vs. red curves). This is due to the glycan-induced delay of head rotation (Figure 2), which ensures the HR1 helix remains directed towards the host as the FPs sample extended configurations. (D–F) The probability that 1, 2, or 3 FPs exceed d_host. In all cases, the presence of glycans shifts the distribution to larger values of d_host, indicating an increased probability of capturing the host. This reveals a critical role for glycans during cell invasion.

Figure 6.. Schematic of fusion mechanism of the Spike protein.

Initial activation of the Spike protein (left) is associated with release of S1, which is triggered by cleavage at the S2’ site and ACE2 receptor binding. When glycans are present (top), HG will enter a caged ensemble where the FPs search for, and capture, the host membrane. HG can then escape the cage, which draws the viral and host membranes together and leads to fusion. In the absence of glycans (bottom), HG can bypass the caged ensemble, resulting in a failed attempt to recruit the host.

Appendix 1—figure 1.. Definitions of domains within the S2 protein.

(A) Prefusion S2 subunit structure of the Spike protein. (B) Postfusion S2 subunit structure of the Spike protein. (C) Sequence range of the Head Group (HG), Fusion Peptide (FP), Connecting Region (CR), Heptad Repeat 1 (HR1), Linker Region (LR), Heptad Repeat 2 (HR2), and Transmembrane Region (TM).

Appendix 1—figure 2.. TM tilt angle distributions.

(A) Distribution of TM tilt angles (defined in SI results section 1.1) sampled during simulations when glycans are present. (B) Distribution of TM tilt angles sampled during simulations when glycans are absent.

Appendix 1—figure 3.. Predicted degree of frustration, by residue.

Density of highly frustrated contacts in a 5Å sphere per residue for prefusion (black) and postfusion (red) S2 subunit structures. Dashed line represents the S2’ cleavage site.

Appendix 1—figure 4.. HG rotation.

(A-C) Single time trace of z_head, r_head and the HG principal axis polar angle, θ. (D–G) Snapshots of the orientation of HG, relative to the membrane. During the prefusion-to-postfusion transition, the head rotates from an orientation in which it is pointing toward the membrane, to an orientation where it is pointing away. Structural snapshots illustrate various orientations during the transition.

Appendix 1—figure 5.. Probability distribution when glycans are absent.

Distribution calculated from 1000 independent simulations without glycans.

Appendix 1—figure 6.. Relative influence of glycans on HR2 and HG.

(A) Structural model with only glycans shown on HG. (B) Structural model with only HR2 glycans present. (C) Distribution of timescales with only HG glycans present. (D) Distribution with only HR2 glycans present. (E) Probability that $d_{\mathrm{FP}}>d_{\mathrm{host}}$ for at least one FP. There is a higher probability of extending to d_host when only HG glycans are present than when only HR2 glycans are present (black vs. red curves).

Appendix 1—figure 7.. Glycans promote host capture with dissociated TM region.

(A-D) Even in the case where the TM strands are able to dissociate, the presence of the glycans increases the probability that the FPs will capture the host membrane. 1000 transitions were simulated for each system. (E–F) Snapshots of glycosylated Spike protein where the TM strands dissociate from one another (glycans are not shown).

Appendix 1—figure 8.. Comparison of probability of FP capture with, and without, a host membrane potential.

Probability of capture, calculated from 1000 simulated transitions. (A) Model with no host membrane, using $d_{host}=27$ to define capture. (B) Model with host membrane potential as defined in Equation S1, which traps (i.e. potential begins to decrease) FPs at ∼27 nm from the virtual viral membrane. Both (A) and (B) show approximately the same probability of 0 FPs being captured, while (B) shows a drastic increase on the probability that capture will involve all three FPs.

Appendix 1—figure 9.. Effective potential for TM confinement in a virtual viral membrane.

The flat-bottom region represents the virtual membrane of width ${\displaystyle w_m=5}$ nm, this region allows the TM motif to move freely between the planes $z=\frac{w_m}{2}$ and $z=\frac{-w_m}{2}$, beyond the flat region an energetic penalty is included to restrain the TM to escape the virtual membrane. Energy is measured in reduced energy units.