Molecular mechanisms of catalytic inhibition for active site mutations in glucose-6-phosphatase catalytic subunit 1 linked to glycogen storage disease

Abstract

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein G6PC1 regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate within the lumen of the endoplasmic reticulum. Because G6PC1 function is essential for blood glucose homeostasis, inactivating mutations cause glycogen storage disease (GSD) type 1a, which is characterized by 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 within the active site as well as explore the energetic perturbations imposed by disease-linked variants. From over 15 μs of MD simulations, 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 that stabilize G6P in the active site. Introduction of GSD type 1a mutations into the G6PC1 sequence causes changes in G6P binding energy, thermodynamic stability and structural properties, suggesting multiple mechanisms 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 side chains.

Identification of structural motifs critical for human G6PC2 function informed by sequence analysis and an AlphaFold2-predicted model

“…The benefit of additional charged residues in the substrate cavity may also explain why individual mutation of residues 36 and 252, which would break the predicted ionic interaction and make another charged residue available for interacting with the substrate, increase the G6Pase activity of G6PC2 whereas mutation of both residues does not ( Figure 4 ). Alternatively, studies in G6PC1 have suggested that D254, the residue corresponding to D252 in G6PC2, may directly interact with G6P during hydrolysis and support the formation of a substrate bound state [ 61 ]. By promoting a potential ionic interaction between residues 36 and 308 in G6PC2, this may allow D252 to interact with the substrate.…”