Mapping Active-Site Conformational Ensembles Along Competing Catalytic Pathways of the Hairpin Ribozyme.

Area of Science:

• Biochemistry
• Molecular Biology
• Computational Chemistry

Background:

• The hairpin ribozyme's catalytic mechanism remains debated, with experimental data supporting multiple interpretations.
• Understanding the precise reaction pathway is crucial for elucidating ribozyme function and designing artificial catalysts.

Purpose of the Study:

• To investigate the conformational landscape of the hairpin ribozyme active site across proposed reaction pathways.
• To differentiate between proposed catalytic mechanisms using advanced computational methods.

Main Methods:

• All-atom molecular dynamics simulations in explicit solvent.
• Enhanced sampling techniques, specifically Hamiltonian replica exchange simulations.
• Exploration of active-site conformations without predefined collective variables for multiple protonation states.

Main Results:

• Dianionic pathways involving A38 and G8 showed distorted active-site geometries, hindering catalysis, especially G8 deprotonation at neutral pH.
• Monoanionic pathways, utilizing scissile phosphate non-bridging oxygens as proton relays, yielded favorable geometries for key reaction steps.
• These monoanionic pathways are compatible with acid catalysis by A38+ and a structural role for G8, without requiring G8's direct catalytic involvement.

Conclusions:

• The study favors monoanionic pathways for hairpin ribozyme catalysis, offering a unified conformational perspective.
• Generated conformational ensembles provide a basis for future QM/MM and ML/MM studies to resolve free-energy landscapes.
• The simulation strategy is applicable to other conformationally flexible biomolecular systems, including other ribozymes.