Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations

Abstract

Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas GTP promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and non-assembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.

FIGURE 1:

Identification of filament disrupting and promoting IMPDH2 mutants. (A) The human IMPDH2 octamer (PDB file 1NF7). One octamer (greens, catalytic domains; pinks, Bateman domains) is shown with a subset of crystal packing neighbors (gray). Tyrosine 12 (red) and arginine 356 (orange) are indicated. (B) Immunoblot of HEK293 cell lysates with antibodies against myc-tagged IMPDH2, total IMPDH, or loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). NT, not transfected. (C) Anti-myc immunofluorescence of HEK293 cells transfected with the indicated IMPDH2-myc constructs (red). 4’6-diamidino-2-phenylindole (DAPI) staining in blue. Cells were either treated with 10 µM mycophenolic acid (MPA) to induce IMPDH2 filaments (arrowheads) or not, as indicated. Images are representative of three experiments. Scale bar, 20 µm. (D) Quantification of transfected cells with filaments; n = 100 cells per sample. (E) Negative-stain electron microscopy of purified IMPDH2 proteins incubated with 5 mM NAD⁺, with and without 1 mM ATP. Images represent two separate experiments. Scale bar, 50 nm. (F) Evolutionary conservation of tyrosine 12 and arginine 356. IMPDH amino acid sequences from Homo sapiens, Xenopus laevis, Callorhinchus milii, Ciona savignyi, and Branchiostoma belcheri were retrieved and aligned using Consurf (Ashkenazy et al., 2016).

FIGURE 2:

IMPDH2 polymerization does not alter its catalytic activity in vitro. (A) Schematic of the IMPDH reaction. (B) NADH fluorescence was monitored in reactions lacking NAD⁺, IMP, IMPDH2 (no enzyme), or a reaction containing all components (Complete). (C) Wild-type and mutant IMPDH2 activity under nonassembling conditions. All mutants showed no significant difference from wild type (two-sided Student’s t test; p > 0.05). Bars denote mean and SD. (D) IMPDH activity assays as in C were conducted in the absence or presence of ATP to induce assembly. ATP did not significantly alter activity in any case (two-sided Student’s t test). (E, F) Initial reaction rates are plotted as a function of substrate concentration for IMP or NAD⁺ comparing wild-type and mutant IMPDH2 under polymerization conditions (1 mM ATP) or for wild type in the presence or absence of ATP (G, H). Michaelis constants determined from these titrations are given in Supplemental Table 1. Two to five replicates per substrate concentration.

FIGURE 3:

IMPDH filament assembly does not alter guanine nucleotide biosynthesis. (A) HEK293 cells transfected with the indicated constructs were visualized with antibodies against IMPDH2-myc, total IMPDH, or a DNA stain (DRAQ5). S275L assembles into filaments while Y12A remains diffusely cytoplasmic. One sample was treated with the IMPDH inhibitor ribavirin. (B) IMPDH immunoblot showing endogenous protein expression (lower band) and myc-tagged IMPDH2 (upper band). (C) Incorporation of [¹³C₂ ¹⁵N]glycine into AMP and GMP. Bars denote mean and standard error for three biological replicates conducted on different days. No statistical differences (two-sided Student’s t test; p > 0.05) were found between samples for incorporation of [¹³C₂, ¹⁵N]glycine into AMP pools. (D) Immunofluorescence localization of myc-IMPDH2 (red) and DAPI (blue). (E) Representative IMPDH or GAPDH immunoblot. (F) [¹³C₂ ¹⁵N]glycine incorporation into AMP and GMP as in C. No statistically significant differences were found (two-sided Student’s t test; p > 0.05).

FIGURE 4:

Ligands influence IMPDH filament architecture by altering the protomer conformation. (A) Representative class averages of IMPDH filaments in four different conformational states. The height of one octamer is indicated. (B) Quantification of the fraction of IMPDH helical segments in each conformational state as a function of ligand concentration. Number of particles counted for each condition is shown. (C) Negative-stain EM reconstruction of IMPDH2 filaments in the expanded, substrate-bound (0.1 mM ATP, 2 mM NAD⁺) and collapsed, GTP-bound (0.1 mM ATP, 0.1 mM GTP) conformations. The refined helical rotation and rise for the expanded filament are 30° and 111 Å and for the collapsed filament 35.5° and 94 Å. (D) The heights of crystal structures of Pseudomonas aeruginosa IMPDH-ATP and Ashbya gossypii IMPDH-GDP closely match the refined helical rise of the human IMPDH2 filaments in the expanded and collapsed states, respectively.

FIGURE 5:

GTP-bound human IMPDH2 adopts the collapsed, inhibited conformation. (A) Cryo-EM reconstruction of Y12A in the presence of 0.1 mM ATP and GTP at 8.7 Å resolution. Catalytic (greens) and Bateman (pinks) domains are well resolved. (B) Atomic model of IMPDH2-ATP-GTP, generated by fitting the catalytic domain and Bateman domains into the cryo-EM structure as two separate rigid bodies. (C) Close-up view of a single IMPDH monomer fitted in the cryo-EM structure. (D) A monomer of the A. gossypii IMPDH-GDP crystal structure (gray) (PDB ID 4Z87) aligned to the human IMPDH2-ATP-GTP model (color) or to the P. aueriginosa IMPDH-ATP monomer (E). (F) Dose-dependent inhibition of wild-type and mutant IMPDH2 by GTP. IMPDH2 was incubated with 1 mM ATP and 3 mM IMP to promote polymerization and then GTP was added for 10 min prior to reaction initiation by with 5 mM NAD⁺. Mean values are plotted and fitted to a three-parameter dose–response curve. Each data point represents three to six replicate reactions. Ninety-five-percent confidence intervals are plotted as dotted lines of the corresponding color. (G) Model: human IMPDH exists in a conformational equilibrium between an expanded, active conformation and a collapsed, inactive conformation that can be shifted by binding to substrates or GTP. Unlike other metabolic filaments, the conformational equilibrium of IMPDH is unaffected by polymerization because the filament form can accommodate both active and inactive conformations.