A Hotspot for Disease-Associated Variants of Human PGM1 Is Associated with Impaired Ligand Binding and Loop Dynamics

Abstract

Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, catalyzing the conversion of glucose 1-phosphate and glucose 6-phosphate. Recently, missense variants of this enzyme were identified as causing an inborn error of metabolism, PGM1 deficiency, with features of a glycogen storage disease and a congenital disorder of glycosylation. Previous studies of selected PGM1 variants have revealed various mechanisms for enzyme dysfunction, including regions of structural disorder and side-chain rearrangements within the active site. Here, we examine variants within a substrate-binding loop in domain 4 (D4) of PGM1 that cause extreme impairment of activity. Biochemical, structural, and computational studies demonstrate multiple detrimental impacts resulting from these variants, including loss of conserved ligand-binding interactions and reduced mobility of the D4 loop, due to perturbation of its conformational ensemble. These potentially synergistic effects make this conserved ligand-binding loop a hotspot for disease-related variants in PGM1 and related enzymes.

Keywords: X-ray crystallography; conformational ensemble; enzyme; inherited disease; loop mobility; missense variants; molecular dynamics.

Figure 1.. Overview of the mechanism and structure of human PGM1.

(A) A schematic of the catalytic reaction, showing the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Glucose 1,6-bisphosphate undergoes a 180° reorientation in between the two phosphoryl transfer steps of the reaction (gray line indicates axis of rotation). (B) The crystal structure of WT human PGM1. Ser117, Arg503 and Arg515 are highlighted as sticks; bound metal ion is shown as black sphere. The missing residues in the D4 loop are shown with dashed line. (C) A close-up of the active site of PGM1. The bound sulfate ion in WT enzyme that acts as a structural mimic for the phosphate group of the substrate is shown with spheres; bound glucose 6-phosphate (in yellow, this report) is superimposed. The Mg²⁺ ion near the site of phosphoryl transfer is shown for reference. Missing residues (507–509) in the D4 loop are shown with dashed line.

Figure 2.. Crystal structures of missense variants of Arg503 and Arg515.

(A) Backbone superposition of WT PGM1 (gray) with R503Q (red), R515L (gold), R515W (purple), R515Q (cyan), and the enzyme-substrate complex with G6P (green). (B) A close-up view of the D4 loop of each structure in (A). The break in the WT chain is indicated with spheres and highlighted by arrows. 2F_o-F_c electron density maps calculated from the final models and contoured at 1.0 σ for the D4 loop in (C) WT PGM1, and the (D) R503Q and (E) R515Q variants. For omit maps, see Figure S2B.

Figure 3.. Crystal structure of the PGM1 complex with glucose 6-phosphate.

(A) A backbone superposition of the apo-enzyme (gray) and enzyme-ligand complex (green). G6P is shown in a space-filling model. (B) Space-filling representation of the PGM1-G6P complex, looking down into the active site. View is a 90° rotation relative to (A). Residues in the mobile flap are in magenta. (C) A close-up view of the PGM1-G6P interactions involving residues in the D4 loop (dashed orange lines). An additional interaction with the backbone amide of Gly506 is not shown. Ser117 and the bound metal ion (black sphere) are shown for reference. For an omit map of the ligand, see Figure S2B.

Figure 4.. Analyses of D4 loop flexibility based on the MD simulations of human PGM1.

(A) RMSF values for WT enzyme mapped onto its structure. Increasing tube radius and a change from blue to red indicate higher RMSF values. (B) A sampling of the D4 loop conformers from the MD trajectory (one structure shown per 10 ps cycle) of WT PGM1. Sampled conformers (white) are shown only for the D4 loop; other regions of the polypeptide backbone are from the crystal structures of the missense variants and PGM1-G6P complex. Colors are as in Figure 2. View is 180° rotation relative to (A). Residence density analysis of the MD trajectories for the D4 loop (residues 505–513) of WT PGM1 (C) and the R503Q variant (D). Maps were calculated as described in Methods. Note the discontinuity of the density for WT D4 loop (blue) versus the continuous density for the R503Q loop (orange).

Figure 5.. Sequences of the D4 loop of phosphoglucomutase in diverse eukaryotic organisms.

Top: a multiple sequence alignment (spanning residues 503 to 515 of human PGM) highlighting identical residues with red background. Bottom: A consensus Web Logo (Crooks et al., 2004) of the D4 loop. R503 and R515 are highlighted by yellow star; variants relevant to this study are indicated with arrows at bottom. Those with confirmed roles in PGM1 deficiency are marked with an asterisk.