Thus, our models were converted back to the WT sequence, as well as cleaved at the S1/S2 interface furin site (R685|S686) to better mimic the expected state in situ. These variants are easier to image and handle experimentally, but these substitutions also potentially alter (23,36) the structure and dynamics of the spike. These models are based on cryo-EM structures (the “experimental structures” below) of the soluble ectodomain of the spike (6VXX, closed (27) 6VSB, chain A open (26)), which included amino acid substitutions (e.g., in 6VSB: R682S, R683G, R685S, K986P, and V987P) to stabilize the spike in the prefusion state. The full-length, glycosylated wild-type spike ectodomain model was described by Casalino et al., (32) in both the closed and 1-up RBD states (the “initial models” below).
All amino acid position numbers refer to those in the full-length spike sequence.
#BULOW DAMAGED VOL 1 ZIP FREE#
The imshow() and contour() functions in the pyplot module of the matplotlib library (59) were used to visualize the free energy landscapes. Backbone RMSD values used Cα, C, and N atoms. (57) RMSD values, CoM angle and dihedral values, and other structural measurements were calculated using the cpptraj (58) module of Amber. (54) Structure visualization and salt bridge identification were performed with VMD. Simulations were carried out using the sander and pmemd. (52,53) Unless otherwise specified, all simulations used default settings in Amber v20, (54) with a 4 fs time step via hydrogen mass repartitioning, (55) an 8.0 Å direct space cutoff with particle mesh Ewald (56) for long-range electrostatics, a Langevin thermostat with a collision frequency of 1.0 ps –1, a Berendsen barostat with a pressure coupling constant of 0.5 ps, and SHAKE on all bonds involving hydrogen atoms with a 0.00001 A tolerance. Simulations described here used the ff14Sbonlysc, (49) GLYCAM, (50) and OPC3 (51) force fields for the protein, glycans, and water, respectively, with salt described by the Joung and Cheatham monovalent ion set. In particular, hinge pocket binders could serve as valuable probes to characterize a stable spike construct over a range of RBD opening angles, providing insight into the coupling of RBD positioning to spike activation. Such hinge pocket binders could avoid problems with low yields (37) that often accompany spike variants that modulate RBD dynamics. These results help rationalize experimental observations (47,48) for the A522V clinical variant and also suggest that binding of a small molecule that makes appropriate contacts in the allosteric hinge pocket could preferentially stabilize the spike in a more open conformation. The pocket is tightly packed when the RBD is closed substitution of A522 with bulkier Val or Leu also remodels the simulated free energy landscape to favor the open, exposed RBD. The conserved K528 forms a salt bridge with D389 only when the RBD is closed, (27) and simulation of K528A shifts the RBD equilibrium toward the open, exposed state. Consistent with experiments, (7,23) the closed RBD is favored on free energy landscapes of the wild-type spike. We calculated free energy landscapes to quantify the role in RBD positioning of specific interactions near the hinge region. In addition to potential value as experimental probes to quantify RBD conformational heterogeneity, small molecules that modulate the RBD equilibrium could help explore the relationship between RBD opening and S1 shedding. Our results also suggest the possibility of allosteric targeting of the RBD equilibrium to favor open states via binding of small molecules to the hinge pocket.
The results provide an explanation for experimental observation of increased antibody binding for a clinical variant with a substitution in this pocket. Using atomistic simulations of the glycosylated wild-type spike in the closed and 1-up RBD conformations, we map the free energy landscape for RBD opening and identify interactions in an allosteric pocket that influence RBD dynamics. The receptor binding domain (RBD) of the spike samples multiple conformations in a compromise between evading immune recognition and searching for the host-cell surface receptor. The spike is a class I viral fusion glycoprotein that extends from the viral surface and is responsible for viral entry into the host cell and is the primary target of neutralizing antibodies. The SARS-CoV-2 coronavirus is an enveloped, positive-sense single-stranded RNA virus that is responsible for the COVID-19 pandemic.
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