Asp263 missense variants perturb the active site of human phosphoglucomutase 1

Abstract

The enzyme phosphoglucomutase 1 (PGM1) plays a central role in glucose homeostasis. Clinical studies have identified mutations in human PGM1 as the cause of PGM1 deficiency, an inherited metabolic disease. One residue, Asp263, has two known variants associated with disease: D263G and D263Y. Biochemical studies have shown that these mutants are soluble and well folded, but have significant catalytic impairment. To better understand this catalytic defect, we determined crystal structures of these two missense variants, both of which reveal a similar and indirect structural change due to the loss of a conserved salt bridge between Asp263 and Arg293. The arginine reorients into the active site, making interactions with residues responsible for substrate binding. Biochemical studies also show that the catalytic phosphoserine of the missense variants is more stable to hydrolysis relative to wild-type enzyme. The structural perturbation resulting from mutation of this single amino acid reveals the molecular mechanism underlying PGM1 deficiency in these missense variants.

Database: Structural data are available in the PDB under the accession numbers 5JN5 and 5TR2.

Keywords: X-ray crystallography; inherited disease; missense variants; phosphoglucomutase; phosphoryl transfer.

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. The glucose 1,6-bisphosphate intermediate undergoes a 180° reorientation in between the two phosphoryl transfer steps of the reaction (gray line indicates axis of rotation). (B) A superposition showing the overall similarity between the structures of WT human PGM1 and the D263G and D263Y missense variants. The WT structure is shown in cyan, D263G in orange, and the D263Y in yellow. The bound metal ion as shown as a magenta sphere and the side chain of Tyr263 with purple spheres.

Figure 2. The context of residue 263 in the structure of WT PGM1

(A) Direct and water-mediated contacts made by the side chain of Asp263, including those with Arg293. Ser117 and the metal ion are shown for reference. WT human PGM1 (PDB ID 5EPC; cyan) is shown in its dephospho-state; the phospho-enzyme version of rabbit PGM (PDB ID 3PMG; 97% identical to human enzyme) is shown in green. Contacts made between Asp263, Arg293, and Ser117 are shown by dashed lines in green for those unique to 3PMG, in blue for 5EPC, and in black when common to both structures. (B) A superposition of related enzymes with PGM1 (cyan) showing the conservation of the analogous aspartate – arginine residue pair in enzymes from different sub-groups and diverse organisms within the superfamily. Structures are from P. aeruginosa phosphomannomutase/phosphoglucomutase (PDB ID 1P5D) in green; Salmonella typhimurium PGM (PDB ID 3NA5) in magenta; parafusin from Paramecium tetraaurelia (PDB ID 1KFI) in gray; and Candida albicans N-acetylglucosamine-phosphate mutase (PDB ID 2DKA) in orange. Ser117 of human PGM1 is shown for reference.

Figure 3. Structural context of residue 263 in the missense variants of PGM1 (clockwise from upper left)

(A) The vicinity of residue 263 in the WT PGM1 (cyan), D263G (orange), and D263Y structures (yellow). Conformational changes in the side chains of His118, His261, Tyr268, and Arg293 are observed relative to the WT enzyme. (B) A close-up view of Arg293 in the D263G and D263Y structures (yellow and orange, respectively) showing its distinct conformation relative to WT enzyme. For comparison, the position of Arg293 in the both phospho- (green) and dephospho-versions (cyan) of WT enzyme is shown in thin sticks (see blue arrow). The loss of the conserved interaction between Asp263 and Arg293 causes the arginine side chain to adopt a novel conformation that makes contacts (dashed lines) with two key ligand-binding residues, Glu376 and Ser378. A water that makes a bridging interaction between Arg293 and Glu376 is shown in red. For clarity, side chains are shown only for D263Y, except for Arg293 where both variants are shown. (C) A model of bound glucose 1-phosphate (green) superimposed with the structure of D263Y (yellow). Arg293 is highlighted with a dotted surface, showing potential steric conflicts with the binding of substrate. The glutamate and serine are highly conserved in the enzyme superfamily, suggesting similar ligand-binding roles in PGM1. The model is based on a superposition of an enzyme-ligand complex (green) from a related protein (PDB ID 1P5D); contacts between G1P and the protein are indicated by dashed orange lines.

Figure 4. Biochemical data on phosphorylation and protein expression/solubility

(A) Time course of dephosphorylation from hydrolysis for WT PGM1 (*, dashed line) and the D263Y (circles, solid line) and D263G (triangles, dotted line) missense variants as determined by ESI-MS. (B) Reduced expression and solubility of the R293A mutant relative to WT PGM1. SDS/PAGE (top) and corresponding histogram (bottom) showing the soluble (yellow) and insoluble (purple) fractions of cell extracts from E. coli cultures of WT PGM1 and the R293A mutant. Relative protein levels on the histogram are normalized to a value of 1.0 using the combined amounts of soluble/insoluble protein obtained for WT PGM1.