Fig. 5

Role of PGAM5 filaments in catalysis. a, b Overview of the a crystal packing in the ∆90 PGAM5 H105A/MM structure (left panel) highlighting a network of stacked rings (right panel), which resembles the organization of b filaments observed for ∆48 PGAM5 in solution by negative-stain EM. c Overview of the ring stacking interface highlighting the positions of residues at the surface of the substrate-binding cleft relative to structural elements contributing to peripheral interactions between two adjacent dodecamers within the crystal lattice of the ∆90 PGAM5 H105A/MM structure. d Electrostatic surface potential calculated for the ∆90 PGAM5 H105A/MM dodecamer using APBS⁷¹. The positions of the MM outlined in green are shown relative to the positions of basic residues R269E and R288E within the positively charged substrate-binding cleft as well as the catalytic residue, H105, in the active site. Coloring corresponds to the electronegativity of the surface potential as defined in the scale bar, with more negatively charged surfaces colored red and positively charged surfaces colored blue. e Upper panel: size exclusion chromatograms of the WT ∆48 PGAM5 purified in buffer containing low (150 mM NaCl) or high (750 mM NaCl) salt concentrations compared to the results obtained for the R269E and R288E variants. Lower panel: EM micrographs of the negatively stained samples of mutant ∆48 PGAM5 taken from the corresponding primary peak of the purification. f Phosphatase activity of the wild type (WT), catalytically inactive H105A, and the stacking-impaired R288E variants of ∆48 PGAM5 measured for 20 nM enzyme and varying concentrations of a phosphorylated ASK1 substrate peptide. Data are represented as mean ± S.E.M. The kinetic parameters K_m, k_cat, and k_cat/K_m were determined using GraphPad Prism and are summarized in Table 2. Scale bars in b and e correspond to 50 nm, except for the inset in b in which the scale bar corresponds to 10 nm