Mapping active-site conformational ensembles along competing catalytic pathways of the hairpin ribozyme

The catalytic mechanism of the hairpin ribozyme has remained controversial for more than two decades, with different experimental approaches often supporting distinct mechanistic interpretations. In this work, we investigate the conformational landscape of the active site along several proposed reaction pathways using all-atom molecular dynamics simulations in explicit solvent combined with enhanced sampling techniques. Specifically, we employ Hamiltonian replica exchange simulations to extensively explore active-site conformations without relying on predefined collective variables, enabling a broad characterization of the structural ensembles associated with multiple protonation states along three candidate reaction mechanisms. Our simulations suggest that a dianionic general acid/general base pathway involving direct participation of A38 and G8 is unlikely to proceed through well-defined intermediates with catalytically competent geometries. In particular, states associated with G8 deprotonation and subsequent O2' deprotonation exhibit strongly distorted active-site arrangements that appear poorly suited for reaction progression. Although highly synchronous proton-transfer steps cannot be excluded, the required deprotonation of G8 remains difficult to reconcile with neutral pH conditions. In contrast, monoanionic pathways in which the nonbridging oxygens of the scissile phosphate act as transient proton relays produce intermediates that sample geometries favorable for the nucleophilic addition and leaving-group elimination steps of the reaction. These mechanisms do not require direct catalytic involvement of G8 while remaining compatible with potential acid catalysis by protonated A38⁺ and a possible structural role of G8. Our results provide a unified conformational perspective on competing mechanistic scenarios. The ensembles generated here offer a foundation for future quantum-mechanical/molecular-mechanical or machine-learning/molecular-mechanics calculations aimed at quantitatively resolving the free-energy landscapes governing hairpin ribozyme catalysis. Finally, the present strategy could easily be applied to other biomolecular systems with high conformational plasticity, including other ribozymes.