We analyzed the spike protein S1/S2 cleavage of selected strains of a prototype coronavirus, mouse hepatitis
virus (MHV) by the cellular protease furin, in order to understand the structural requirements underlying the
sequence selectivity of the scissile segment. The probability of cleavage of selected MHV strains was first evaluated
from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured
experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin.
We found that in vitro cleavability varied across MHV strains in line with predicted results—but with the notable
exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known
X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried
out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site,
and the suitability of the complex for initiation of the enzymatic cleavage. We identified the 3D structural requirements
of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in
which the various strains tested experimentally are fulfilling these requirements. We find that despite some
flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at
P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces
a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements
for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the
conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by
arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the
function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the
spike S1/S2 structural loop.
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Cooperative multivalent receptor binding promotes exposure of the SARS-CoV-2 fusion machinery core
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.
more » « less- Award ID(s):
- 2029092
- NSF-PAR ID:
- 10363256
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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