Hawes, E. M. | bioRxiv Science

bioRxiv2026

Sequence determinants of pathogenicity in glucose-6-phosphatase linked to glycogen storage disease type 1a

Glycogen storage disease type 1a (GSD1a) is an autosomal recessive Mendelian disorder that can be caused by missense variants in glucose-6-phosphatase catalytic subunit 1 (G6PC1). Although hundreds of missense variants have been identified, the vast majority are of unknown clinical significance, and the molecular mechanism(s) of bona fide pathogenic variants are ill-defined. We combine bioinformatic data and clinical associations with the protein language model AlphaMissense to guide mechanistic exploration of 78 missense variants at 55 residue positions using robust biochemical and biophysical assays to distill general principles of enzyme dysfunction. Correlation analysis established a strong linear relationship between folded G6PC1 abundance and catalytic capacity for most variants. Pathogenic variants within this paradigm were linked to compromised stability and activation of the unfolded protein response. However, outliers characterized by relatively high abundance, yet low activity clustered to a network of sidechains adjacent to the active site that allosterically modulate catalysis. Contextualized by recent high-resolution structures and AlphaFold modeling, our holistic analysis of G6PC1 in vitro metrics facilitates clinical (re)classification of variants according to explicit molecular phenotypes and identifies therapeutic design directions. Moreover, our approach illustrates a blueprint for variant characterization that integrates computational prediction with experimental validation to discover disease etiology.

biochemistry↗

bioRxiv2023

Molecular mechanisms of functional impairment for active site mutations in glucose-6-phosphatase catalytic subunit 1 linked to glycogen storage disease type 1a.

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein 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 cause 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. Exploiting a computational model of G6PC1 derived from the groundbreaking structure prediction algorithm AlphaFold2 (AF2), we combine molecular dynamics (MD) simulations and computational predictions of thermodynamic stability with a robust in vitro screening platform to define the atomic interactions governing G6P binding as well as explore the energetic perturbations imposed by disease-linked variants. 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. Introduction of GSD type 1a mutations into the G6PC1 sequence elicits changes in G6P binding energy, thermostability and structural properties, suggesting multiple pathways of catalytic impairment. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm active site structural organization but also suggest novel mechanistic contributions of catalytic and non-catalytic side chains.

biophysics↗