Free-Energy Landscapes of HBV Hexamer Closure Reveal Key Structural Features of the Transition.

Chronic infection with the hepatitis B virus (HBV) remains a major global health burden and depends on efficient viral replication, including the assembly of the viral capsid during the HBV life cycle. Capsid assembly proceeds through transient oligomeric intermediates, among which the formation of hexameric units is thought to underlie the energetic bottleneck associated with nucleation during capsid assembly. Despite extensive experimental and computational work, the structural and energetic determinants of hexamer closure remain incompletely understood at the molecular level. Here, we employ a multistage computational approach to investigate the open-to-closed transition of the HBV capsid hexamer in the apo system, in which targeted molecular dynamics is employed to generate diverse open-to-closed transition pathways. These pathways are subsequently refined and sampled by using path-based free-energy methods to construct multidimensional free-energy landscapes of hexamer closure. Across independently sampled pathways, we observe diverse transition routes, while a conserved steric rearrangement in the gate region emerges as the dominant rate-limiting feature. These results provide a qualitative characterization of hexamer closure energetics and establish a general framework for studying complex conformational transitions in large, flexible biomolecular assemblies.