Simulations reveal the role of glucose-6-phosphatase mutations in glycogen storage disease.

Abstract

Glucose-6-phosphatase catalytic subunit 1 (G6PC1) is a key enzyme in central metabolism, specifically in gluconeogenesis and glycogenolysis, but its structure and dynamics have eluded the scientific community for decades. Particular mutations in this enzyme give rise to glycogen storage disease type 1a, making it a potential therapeutic target. Recent advances in protein structure prediction by AlphaFold2 have yielded the first structural model of G6PC1. In a dual-pronged computational and experimental approach, we have investigated the structural and dynamical properties that confer G6PC1 catalytic activity. Multiple replica equilibrium molecular dynamics (MD) simulations allowed us to obtain a relaxed consensus structure of G6PC1 in a representative endoplasmic reticulum lipid bilayer. We then obtained a liganded structure of G6PC1 for both alpha and beta isomers of its native substrate, glucose-6-phosphate, by leveraging docking data and homologous crystallographic information. To assess the role of mutations on ligand stability, we collected over 15 μs of MD simulation of wildtype and clinically relevant mutants. From this data we identified novel substrate-interacting residues by hydrogen bonding networks which we show have destabilizing effects on protein-ligand interactions, quantified by free energy calculations. Our expression and activity assays further corroborate the robustness of these wildtype and mutant models. This study provides the most detailed dynamical description of substrate-bound G6PC1 and elucidates the role of activity perturbing mutants on its structure, energetics, expression and activity. Together these findings suggest a molecular mechanism by which G6PC1 mutations may propagate clinical phenotypes of glycogen storage disease.