Cooperative multivalent receptor binding promotes exposure of the SARS-CoV-2 fusion machinery core

Abstract

The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes dissociation of the S1 domains and exposure of the fusion machinery, although the molecular details of this process have yet to be observed. We report the development of bottom-up coarse-grained (CG) models consistent with cryo-electron tomography data, and the use of CG molecular dynamics simulations to investigate viral binding and S2 core exposure. We show that spike trimers cooperatively bind to multiple ACE2 dimers at virion-cell interfaces in a manner distinct from binding between soluble proteins, which processively induces S1 dissociation. We also simulate possible variant behavior using perturbed CG models, and find that ACE2-induced S1 dissociation is primarily sensitive to conformational state populations and the extent of S1/S2 cleavage, rather than ACE2 binding affinity. These simulations reveal an important concerted interaction between spike trimers and ACE2 dimers that primes the virus for membrane fusion and entry.

Fig. 1. Schematic overview and characterization of our coarse-grained (CG) models.

a Representative depiction of a CG simulation of spike trimers in membrane interacting with an adjacent membrane with ACE2 dimers. The insets depict the CG model components for the spike trimer (bottom), ACE2 dimer (upper left), and lipid membrane (upper right). Note that the protein CG sites are colored by monomer while glycans are represented by gray balls. b Probability distributions for RBD conformations projected onto the first time-lagged independent component (tIC₁). The purple, gray, and orange distributions (N = 19383, 33810, and 21810, respectively) denote the three clusters identified by k-means clustering with labels indicating their representative conformational state. c Comparison of spike trimer conformational state populations (ranging from 0 to 3 open RBDs or shed RBDs, i.e., exposure of the S2 trimeric core) from CG simulations (N = 53076) and cryo-ET classification (N = 4220); the core exposed state is in the prefusion form in the CG simulations while it is in the postfusion form in the cryo-ET dataset. d Representative depiction of the average configuration of CG S1 (brown beads) within the identified k-means clusters. For reference, CG configurations of experimentally-resolved open and closed states of S1 are shown as magenta and cyan beads, respectively. Arrows indicate the positions of each respective RBDs. In all cases, the N-terminal domains of S1 are aligned.

Fig. 2. Dissociation of S1 facilitated by multivalent ACE2 binding.

(Top) Snapshots of one representative S1 dissociation process upon binding of two ACE2 dimers. Each monomer in the ACE2 dimer is represented by red and blue beads, respectively, while each S1 protomer in the spike trimer is represented by cyan, pink, and green beads, respectively. The gray and silver beads denote glycans and S2 protomers, respectively. The depicted process is also shown in Supplementary Movie 1. (Middle) Time series profile depicting tIC₁ evaluated for the RBD of each protomer (sharing the same color) for the spike trimer depicted above, i.e., spike trimer protomers are labeled cyan to pink to green (back to cyan) in counter-clockwise order when viewing from the top-down. To the right of the panel, the double-ended arrows represent the extent of the tIC₁ distributions for each state from Fig. 1b, while the rectangle shows the peak of each distribution. (Bottom) Time series profile depicting k-means cluster IDs for the RBD of each protomer (same color scheme as the middle panel). To the right of the panel, the structural class assigned to each cluster ID is annotated. Each time series profile depicts the mean (line) and standard deviation (shaded region) of N = 25000 points using block averaging over 500 points.

Fig. 3. Structural changes to S1 upon ACE2 binding.

a Schematic of the four geometric points (purple spheres) in S1 used to define the hinge dihedral and the cavity area. b, c Probability distributions of the hinge dihedral in each of the three structural states (closed, partially closed, and open) in the absence (N = 75003) and presence (N = 43800) of ACE2. d, e Probability distributions of the cavity area in each of the three structural states (closed, partially closed, and open) in the absence (N = 75003) and presence (N = 43800) of ACE2.

Fig. 4. Cooperative enhancement of S2 trimeric core exposure by ACE2.

a Summary statistics for the fraction of S1 monomers bound to ACE2 (blue), S1 monomers dissociated from the spike trimer (red), and spike trimers with complete exposure of the S2 trimeric core (green) as the stoichiometric ratio of ACE2 dimers to spike trimers, [ACE2_dimer]/[S_trimer], is varied. b Hill plot comparing the natural log of the surface density of ACE2 (in #/nm²) to the natural log of the fraction of S1 monomers bound to ACE2 or the fraction of completely exposed S2 trimeric cores (Θ). The black and gray lines are fits to the Hill equation with Hill coefficients (n_H) equal to 1.45 and 2.02. All data points report the mean and standard error from triplicate simulations.

Fig. 5. Comparison of variant-emulating CG models to that of the wild type (WT).

a Summary statistics for the difference in the probabilities of the closed (black), 1 RBD open (red), 2 RBD open (green), and 3 RBD open (blue) conformational states for the variants and that of the WT (P_variant − P_WT). b Summary statistics for the difference in the fraction of S1 monomers bound to ACE2 (blue) and spike trimers with complete exposure of the S2 trimeric core (green) for the variants and that of the WT (f_variant – f_WT); here, the stoichiometric ratio of ACE2 dimers to spike trimers is 2.56:1 (i.e., 64 ACE2 dimers to 25 spike trimers). The five variant-emulating CG models are: enhanced S1–S2 interactions ((+)S1S2), enhanced RBD-ACE2 interactions ((+)RBDACE2), enhanced S1–S2 and RBD-ACE2 interactions ((+)S1S2(+)RBDACE2), increased open-state conformational sampling (E_0.3c+0.7o), and increased closed-state conformational sampling (E_0.7c+0.3o). All data points report the mean and standard error from triplicate simulations.

Fig. 6. Comparison between spike trimer S1/S2 cleavage states for the wild type (WT) CG model.

a Summary statistics for the difference in the probabilities of the closed (black), 1 RBD open (red), 2 RBD open (green), and 3 RBD open (blue) conformational states for the partially cleaved and completely cleaved spike trimer states (P_uncleaved − P_cleaved) for WT virus. b Summary statistics for the difference in the fraction of S1 monomers bound to ACE2 (blue) and spike trimers with complete exposure of the S2 trimeric core (green) for the partially cleaved and completely cleaved spike trimer states (f_uncleaved − f_cleaved) for WT virus; here, the stoichiometric ratio of ACE2 dimers to spike trimers is 2.56:1 (i.e., 64 ACE2 dimers to 25 spike trimers). In each simulation, the cleavage states are homogeneously distributed, e.g., the 66% cleaved system consists of 2 out of 3 cleaved protomers in every spike trimer. All data points report the mean and standard error from triplicate simulations.