SELECTIVE DESTABILIZATION OF ATP BINDING AT CBS3 CAUSED BY THE PRKAG2 H401Q MUTATION IN HUMAN AMPK γ2

Mutations in the PRKAG2 gene, which encodes the γ2 subunit of AMP-activated protein kinase (AMPK), are associated with a rare inherited glycogen-storage cardiomyopathy characterized by ventricular hypertrophy, arrhythmias, and sudden cardiac death. The H401Q variant was identified in a family in which sudden cardiac death was the first clinical manifestation; however, its underlying molecular mechanism remains poorly understood. Since the γ2 subunit regulates AMPK activity through adenine nucleotide sensing, alterations in nucleotide recognition may disrupt enzyme regulation. In this study, we investigated the structural and energetic effects of the PRKAG2 H401Q mutation using homology modeling, molecular dynamics (MD), principal component analysis (PCA), and metadynamics simulations. A structural model of the human AMPK γ2 subunit was generated in AutoModel using the murine γ1 structure (PDB ID: 2V8Q) as a template, with H401Q corresponding to H146Q in the modeled system. Wild-type and mutant γ2 systems underwent triplicate 800 ns MD simulations using GROMACS 2022-6. Nucleotide-binding energetics at functional sites CBS1, CBS3, and CBS4 were investigated using metadynamics simulations with AMP and ATP in Desmond-Maestro 2022-4. Eight independent replicas were performed for each ligand-site condition, with simulation lengths ranging from 60 to 100 ns. MD simulations showed that H401Q preserves overall structural stability while increasing conformational heterogeneity and local flexibility. PCA and RMSF analyses revealed enhanced mobility in the 246-258 loop and additional dynamic changes at the γ-α subunit interface, suggesting that the effects of the mutation extend beyond the nucleotide-binding region. This altered dynamic behavior was accompanied by remodeling of the local hydrogen-bond network, including persistent interactions involving Ser293 and His128. Metadynamics simulations demonstrated that H401Q selectively destabilizes ATP binding at CBS3, shifting the binding free energy from -13.5 kcal/mol in the wild type to -10.0 kcal/mol in the H146Q mutant, while preserving AMP recognition. No significant energetic changes were observed at CBS1 or CBS4. This site-specific allosteric alteration may impair AMPK sensing of elevated cellular energy states (ATP), suggesting a molecular mechanism underlying the metabolic dysregulation associated with PRKAG2 syndrome.