Integrating Parallel-Bias Metadynamics-Metainference and Relaxation Dispersion NMR to Resolve Hidden Conformational...

Conformational dynamics in toxin inhibitors are an important contributor to ion channel affinity, yet toxin multidimensional energy landscapes remain largely unexplored. In the current work, we combine parallel-bias metadynamics-metainference (PBMetaD) simulations with relaxation dispersion NMR to define, at atomistic resolution, the thermodynamics and kinetics of Hui1, a de novo three disulfide toxin derived from the SAK-I family that targets K+-channels. Using the three χ3 disulfide dihedrals as collective variables, an extensive 48-replica well-tempered PBmetaD simulation (16.2 μs cumulative sampling) resulted in a fully converged three-dimensional (3D) free-energy surface comprising eight Hui1 conformers. These basins account for ∼96% of the bias-weighted ensemble and partition into four low- and four high-energy states separated by 7.5 kJ/mol associated with the (-) and (+)Cys12-Cys28 χ3 states, respectively. Transition-state theory identifies rota-isomerization of Cys3-Cys35 as the slowest, and therefore rate-determining, coordinate, while the analogous motions around Cys12-Cys28 and Cys17-Cys32 are ∼5-fold faster. 15N R1ρ relaxation dispersion NMR measurements confirmed these kinetics, identifying two structurally distinct residue clusters exhibiting intermediate and faster exchange processes. The Key Interaction Finder (KIF) approach reveals that Cys17-Cys32 conformation modifies connectivities within a dense interaction network between Cys17 and residues Gln14, Tyr23, Arg24, and Lys29, and correlates with the accessibility of residues Tyr23 and Arg24 of the helix-kink-helix region for interaction with the channel vestibule. Our work establishes PBMetaD as a powerful framework for mapping coupled disulfide and backbone dynamics in toxins and reveals specific conformers and interactions likely to control K+ channel recognition.