Dual-Locking the SARS-CoV-2 Spike Trimer: An Amphipathic Molecular "Bolt" Stabilizes Conserved Druggable Interfaces for Coronavirus Inhibition.

The SARS-CoV-2 spike (S) protein, a trimeric structure comprising three receptor binding domains (RBDs) and three N-terminal domains (NTDs), undergoes substantial conformational changes to a fusion-prone open state for angiotensin-converting enzyme 2 (ACE2) binding and host cell infection. Stabilizing its closed state is a key antiviral strategy but remains challenging. Here, we introduce S416, a novel amphipathic molecule acting as a "molecular bolt". Cryo-EM study reveals that S416 binds concurrently to six sites across two distinct druggable interfaces: three molecules at the RBD-RBD interfaces and three at the NTD-RBD interfaces. This unique "dual-locking" mechanism, driven by S416's polar carboxyl head and nonpolar phenylthiazole tail, robustly stabilizes the spike trimer in a locked, closed conformation through strong inter-domain interactions, reducing structural flexibility and atomic fluctuations compared to the apo structure resolved synchronously. Crucially, these RBD-RBD and NTD-RBD interfaces are conserved across human-infecting coronaviruses, suggesting potential as broad-spectrum antiviral targets. Our findings demonstrate that the highly dynamic spike trimer can be effectively stabilized by an amphipathic molecular bolt targeting both the inter- and intra-monomer interfaces, offering a promising strategy against emerging coronaviruses.