Comparative analysis of AMP and ATP binding affinities in wild-type and PRKAG2-mutant γ2 subunits of AMPK: Insights from Metadynamics simulations.

AMP-activated protein kinase (AMPK) is a crucial heterotrimeric enzyme comprised of a catalytic α subunit and regulatory β and γ subunits. The γ subunit, characterized by four cystathionine-β-synthase (CBS) repeats, features four potential binding sites (sites 1-4) for AMP, ADP, or ATP and plays a crucial role in sensing cellular energy status by responding to fluctuations in AMP/ADP and ATP levels. Mutations in the γ2 subunit, such as the PRKAG2-p.His401Gln mutation is linked to PRKAG2 syndrome, a genetic disorder associated with cardiomyopathy. This study investigates the binding affinities of AMP and ATP to the γ2 subunit of wild-type and PRKAG2-p.His401Gln mutant AMPK. Using a γ2 model of human AMPK, we conducted 60 ns metadynamics simulations with the Desmond software, employing the OPLS-2005 force field. Key variables analyzed included the distance of each ligand from the initial contact zone and the C-N-C-O dihedral angle between the nitrogenous base and ribose. We assessed the free energy changes (ΔG) and visualized the trajectories using Schrödinger Maestro. Our results indicate that, in the mutant structure, ATP at site 3 tends to remain in the initial contact zone, reflecting an energetically unfavorable binding mode. Conversely, in the wild-type structure, ATP prefers to fit the final binding site. Despite these observations, statistical analysis (ANOVA) revealed significant differences in site 3 binding ΔG between the wild-type and mutant complexes. The potential impact of future studies, which will involve essential dynamics experiments with the full-length AMPK protein to further explore the relationship between subunit dynamics, ligand binding, and structural dynamics of human AMPK, is promising and will significantly contribute to our understanding of AMPK function and dysfunction.