Integrative analysis of pathogenic variants in glucose-6-phosphatase based on an AlphaFold2 model

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein glucose-6-phosphate catalytic subunit 1 (G6PC1) regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 causes glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. In this study, we determine the atomic interactions governing G6P binding as well as explore the perturbations imposed by disease-linked missense variants by subjecting an AlphaFold2 G6PC1 structural model to molecular dynamics simulations and in silico predictions of thermodynamic stability validated with robust in vitro and in situ biochemical assays. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. The introduction of GSD type 1a mutations modified the thermodynamic landscape, altered side chain packing and substrate-binding interactions, and induced trapping of catalytic intermediates. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm the active-site structural organization but also identify previously unobserved mechanistic contributions of catalytic and noncatalytic side chains.