Structural dynamics and allosteric communication of a SARS-like bat coronavirus spike glycoprotein

SARS-like bat coronaviruses (CoVs) pose ongoing public health risks due to their zoonotic potential, making it important to understand the molecular pathways that drive their evolution. We recently showed that SHC014-CoV can infect human cell lines in an angiotensin-converting enzyme 2 (ACE2)-dependent manner after acquiring two spike ectodomain mutations (F294L and A835D). However, how the wild-type (WT) SHC014 spike differs dynamically from these mutants remains unclear. Here, we built fully glycosylated ectodomain models of WT and three mutants (F294L, A835D, and the double mutant [DM]) and performed triplicate 1-μs all-atom molecular dynamics (MD) simulations for each variant. The two mutations exhibit epistasis, altering structural rearrangements relative to the WT. Notably, the DM receptor-binding domain (RBD) begins sampling the open conformation in our conventional MD. At the atomic level, the DM spike mitigates the dense negative packing introduced by A835D through a salt-bridge network, while F294L disrupts π-mediated interactions, together enhancing RBD opening propensity, which is critical for viral entry. Increased flexibility of the subdomain-2 "620-loop" further modulates DM RBD openness. Dynamical network analysis identified three allosteric communication pathways. In WT and F294L, "pathway 1" forms the baseline route linking the 620-loop to the RBD, whereas in A835D and the DM, it extends to the fusion peptide proximal region (FPPR), reshaping long-range communication. "Pathway 2" is conserved across variants but is most prominent in WT and F294L. "Pathway 3" appears only in A835D and the DM, compensating for reduced communication along pathway 2. Overall, this work provides an atomistic perspective on SHC014 molecular adaptation during host-to-host transmission and highlights mechanistic features that may inform future therapeutic and pandemic-preparedness efforts.