Structure and Characterization of Phosphoglucomutase 5 from Atlantic and Baltic Herring-An Inactive Enzyme with Intact Substrate Binding

Abstract

Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.

Keywords: adaptation; enzyme structure; herring; phosphoglucomutase 5; phosphohexomutase.

Figure 1

General reaction mechanism of phosphoglucomutase (PGM) enzymes. P = phosphate group.

Figure 2

Strong association between variation at the PGM5 locus in herring and ecological adaptation to the Baltic Sea. (a) Geographic distribution of the population samples of the Atlantic and Baltic herring (1–53) analyzed by whole genome sequencing in Han et al. [5]. X-axis is longitude and Y-axis is latitude. (b) Genome-wide screen for allele frequency differences between the Atlantic and Baltic herring. The P-value for the most significant single nucleotide polymorphisms (SNP) on chromosome 18 is P = 10⁻²⁶⁶. Pie charts are the frequency of the PGM5 Ala330Val substitution. (c,d) Zoom in showing delta allele frequency (frequency in Baltic herring vs. frequency in Atlantic herring); for SNPs on chromosome 18, the location of the PGM5 gene is indicated. (e) Comparison of salinity at each sample location and allele frequencies of the PGM5 Ala330Val missense mutation (rs5164711) and the SNP (rs5199622) showing the most extreme difference in allele frequency between the Atlantic and Baltic herring. Numbers 1–53 refer to sample locations in Figure 2a, and P represents Pacific herring.

Figure 3

Gene architecture of PGM5 in chromosome 18. The top track (red) shows the genomic footprint of the PGM5 gene model, each subsequent track shows the exon–intron organization of one transcript. SNPs of interest are shown by position and ref/alt base.

Figure 4

Apo structures of PGM5. (a) Overall structure of herring PGM5. aPGM5 is colored according to domain (domain I green, domain II pink, domain III orange, and domain IV blue), while bPGM5 apo is shown in semi-transparent gray. The A330V mutation site is shown as red spheres. The root mean square deviation (RMSD) between the two variants is 0.64 Å over 545 C_α atoms. (b) Overlay of the active site of apo aPGM5 (dark colors) and bPGM5 (light colors). Active-site loops are colored according to domain as in A. The phosphorylated active-site Ser121 in bPGM5 is shown as sticks and the active-site metal ions are shown as spheres (green—Ca²⁺ in aPGM5, dark green—Ni²⁺ in bPGM5). (c) Superposition of PGM structures in ribbon based on domains I-III, for clarity no ligands are shown (aPGM5 apo (green), bPGM5 apo (cyan), human PGM1 (PDB 5EPC chain B, light gray; RMSD 1.67 Å over 529 C_α atoms), rabbit PGM1 (PDB 3PMG chain A, dark gray; RMSD 1.58 Å over 481 C_α atoms), parafusin with bound sulfate (PDB 1KFI chain A, yellow; RMSD 1.19 Å over C_α atoms), and human PGM1-2 (PDB 6SNO chain A, purple; RMSD 1.31 Å over 534 C_α atoms)). All RMSD values compared to aPGM5 apo.

Figure 5

Mutation site and differences in crystal contacts. Symmetry-related molecules are shown in gray. Dashed lines indicate hydrogen bonds. (a) Crystal contact of Val330 (red) in bPGM5 (cyan). Side chains within 6Å are shown. (b) Corresponding region of Ala330 (red) in aPGM5 (green). Side chains within 6Å are shown. (c) β-sheet-extending crystal contact involving 7 ordered residues in the N-terminus of bPGM5 (cyan). A Ca²⁺ ion (green sphere) is observed in this interface, interacting with the symmetry-related molecule, and through bridging waters to Glu2 of the N-terminus. (d) Crystal contacts of the N-terminus of aPGM5 (green). Side chains within 6Å are shown.

Figure 6

Structure of bPGM5 in complex with G1P. G1P is shown in magenta as sticks, active-site loops are colored according to domain (loops 0 and 1 in green, loop 2 in pink, loop 3 in yellow, loop 3B in orange, and loop 4 in blue). Ni²⁺ is shown as a green sphere. Smaller purple and red spheres represent Na⁺ and waters. Dashed lines indicate hydrogen bonds and metal interactions. (a) Polder F_o-F_c omit maps (mesh) of G1P (gray), phosphoserine 121 (blue), and Ni²⁺ (green) contoured at 5σ (0.43–0.45 e-/ų). (b) G1P binding site, with G1P, and interacting side chains. (c) Comparison of overall structure of bPGM5 in complex with G1P (orange, G1P in magenta), human PGM1 in complex with G6P (6BJ0, chain B, gray, G6P in green), and human PGM1-2 in complex with G1P (6BJ0, chain B, dark gray, G1P in blue). Ni²⁺ (bPGM5, green), Mg²⁺ (hsPGM1, light green), and Zn²⁺ (hsPGM1-2, gray) are shown as spheres. Structures are aligned based on domains 1–3. (d) Comparison of the binding sites of G1P in bPGM5 (colors as in b) and of G6P in hsPGM1 (6BJ0, chain B, gray with ligand as sticks and Mg²⁺ in green). Interacting side chains within 6Å of ligands are shown as lines with residues displaying differences highlighted as sticks. (e) Comparison of the binding sites of G1P in bPGM5 (colors as in b) and of G1P in hsPGM1 isoform 2 (6SNO, chain A, gray with ligand as sticks and Zn²⁺ in gray). Side chains within 6Å of ligands are shown as lines with residues displaying differences highlighted as sticks.

Figure 7

Logo representation of the sequence conservation of active-site loops (loop 0–4) and the Ala330Val mutation region of PGM1 and PGM5 from different species. The logos are based on sequence alignments of PGM5 and PGM1 from 10 fishes and from 10 mammals (Supplementary Table S5, Figure S8). The top and bottom sequence and numbering are from the Baltic herring and human, with important catalytic residues (phosphorylated serine, catalytic acid, and base) and the mutation site (Val330 and corresponding amino acid) marked in red and bold. The logos were generated using Weblogos [58].

Figure 8

Detailed comparisons of loops 1 and 2 in PGM5 and PGM1. Amino acids are numbered according to herring PGM5. Dashed lines in the same color as the respective structure indicate hydrogen bonds. Mg²⁺ (hPGM1, light green), Ca²⁺ (aPGM5, green), Zn²⁺ (gray, hsPGM1-2), or Ni²⁺ (bPGM5, dark green) ions are shown as spheres. Na⁺ ions in aPGM5 and bPGM5 shown as small light purple or purple spheres. The structures are aligned based on loop1 (a,b) or loop 2 (c). (a) Comparison of loop 1 in apo structures of herring PGM5 and hsPGM1. Overlay of aPGM5 apo (green), bPGM5 apo (cyan), and hsPGM1 apo (PDB 5EPC chain B, gray). In bPGM5 apo, Ser121 is phosphorylated. Sulfate bound in hPGM1 shown as sticks. (b) Comparison of loop 1 in complex structures of herring PGM5 and hsPGM1. Overlay of bPGM5 (orange) and G1P (magenta) with hsPGM1 (PDB 6BJ0 chain B, gray) and G6P (dark gray) and hsPGM1-2 (PDB 6SNO chain A, blue) and G1P (blue). Sidechains are shown as sticks. In bPGM5 and hsPGM1-2, the serine is phosphorylated. G1P and G6P shown as sticks. (c) Comparison of loop 2 in aPGM5 apo (green), bPGM5 apo (cyan), bPGM5 and G1P (orange), human PGM1 apo (PDB 5EPC chain B, light gray), and human PGM1 and G6P (PDB 6BJ0 chain B, dark gray). Sidechains of residues 288–293 in human PGM1 and residues 292–297 in herring PGM5 are shown as lines.