Conformational Dynamics and Disorder of HPV-16 E6

Abstract Persistent infection with high-risk human papillomavirus (HPV) types, notably HPV-16, drives oncogenesis through the multifunctional E6 oncoprotein (16E6), which hijacks host regulatory pathways by engaging diverse protein partners. Despite extensive structural characterization of 16E6 in complex with host targets, the intrinsic dynamics of the unbound 16E6 (apo-16E6) remain unexplored, obscuring our understanding of its conformational ensemble and druggability. Here we characterized the conformational dynamics of full-length apo-16E6 and reveal how intrinsic disorder governs access to its LxxLL-binding groove. We combined exhaustive all-atom molecular dynamics (MD) simulations with state-of-the-art enhanced sampling approaches, including well-tempered metadynamics (WT-MetaD) and replica exchange with solute tempering (REST2), to generate an aggregated 47-μs dataset describing the conformational landscape of apo-16E6. Across methods, the interdomain α-helical linker (E6HL) and the C-terminal tail emerge as intrinsically disordered regions (IDRs) that choreograph large-amplitude motions of E6N and E6C and spontaneous interdomain opening of apo-16E6 from the closed crystal structure. Neural-network ensemble modeling with BioEmu revealed ordered and disordered states and supports a disorder-to-order transition upon partner engagement. Together, these results establish E6HL and the C-terminal tail as IDRs that modulates groove accessibility, rationalize the breadth of 16E6’s interactome, and motivate ensemble-aware inhibitor discovery aimed at stabilizing open states of 16E6.