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IMP dehydrogenase (IMPDH) controls a key regulatory node in purine biosynthesis. Gain-of-function mutations in human IMPDH2 are associated with neurodevelopmental disorders and neuromuscular symptoms including dystonia, but the developmental mechanisms underlying these defects are unknown. We previously showed that these mutants are insensitive to GTP inhibition and hypothesized that their hyperactivity would affect nucleotide metabolism in vivo. Here, we characterize the metabolic and developmental consequences of the neurodevelopmental disorder-associated IMPDH2 mutant, S160del, in Xenopus tropicalis. We show that expressing S160del but not WT human IMPDH2 disrupts purine pools and somite organization in the developing tadpole. We also show that S160del disrupts in vivo IMPDH filament assembly, a well-described IMPDH regulatory mechanism. Cryo-EM structures show that S160del disrupts filament assembly by destabilizing the dimerization of regulatory Bateman domains. Dimerization of Bateman domains and subsequent filament formation can be restored with a high affinity ligand, but this does not restore sensitivity to GTP inhibition, suggesting S160del also disrupts allostery of IMPDH2 filaments. This work demonstrates that the structural effects of patient IMPDH2 variants can cause disruptions both to nucleotide levels and to the normal development of sensorimotor structures, helping us better understand the physiological basis of disease in these patients.
R35GM149542 HHS | NIH | National Institute of General Medical Sciences (NIGMS), 5R01GM148490 HHS | NIH | National Institute of General Medical Sciences (NIGMS), 5R01NS099124 HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS), T32GM008268 HHS | NIH | National Institute of General Medical Sciences (NIGMS), R35 GM149542 NIGMS NIH HHS , T32 GM008268 NIGMS NIH HHS , R01 NS099124 NINDS NIH HHS , R01 GM148490 NIGMS NIH HHS
Fig. 2. Tadpoles expressing hIMPDH2-S160del have somitic defects. (A) Diagrams of experimental design and simplified X. tropicalis tail anatomy. X. tropicalis embryos are injected with 500 pg of mRNA into each cell at the 2-cell stage and then raised to NF stage 41, approximately 3 dpf. (B) Representative anti-neurofilament immunofluorescence images of tadpoles injected with the indicated mRNA. (C) Representative anti-skeletal muscle immunofluorescence images of tadpoles injected with the indicated mRNA. (D) Quantification of the number of intersomitic axon bundles per mm in the tail. One-way ANOVA and Tukey’s multiple comparisons test. (E) Quantification of somite boundary quality. Fisher’s exact test for categorical variables.
Fig. 3. S160del destabilizes IMPDH superstructures in vivo. (A) Experimental diagram. X. tropicalis embryos are injected with 500 pg of mRNA into each cell at the 2-cell stage, raised to NF stage 35 (approximately 2 dpf), and then treated with either 1 uM MPA or vehicle control. Tadpoles are raised for an additional 24 h to NF stage 41 and then stained for IMPDH2. (B–G) Representative anti-IMPDH2 immunofluorescence images of uninjected (endogenous) or mRNA injected tadpoles treated with either 1 μM MPA (B–D) or DMSO vehicle control (E–G). Insets in the top right corners of each image are close-ups of the regions indicated by white boxes.
Fig. 4. S160del destabilizes IMPDH2 polymerization in vitro. (A) Structure of the hIMPDH2 monomer under physiologically relevant ligand conditions (with bound ATP, GTP, IMP, NAD+). Serine 160 is located within the regulatory Bateman domain and is proximal to both ATP in site 1 and GTP in site 2 when bound. Potential hydrogen bonds to the phosphates of ATP in site 1 are displayed as dashed lines. (B) Distribution of S160del assembly states as determined by cryo-EM. Selected 2D class averages are shown. The majority of selected particles (68%) are classified as interfacial octamers with a smaller subset (28%) of tetramers. Only 4% of selected particles existed in the canonical octamer state, but secondary structure is apparent in the Bateman domains. (C and D) Cryo-EM map of the S160del interfacial octamer (C). Only the catalytic domain is resolved (green). The disordered Bateman domain is represented as a purple dashed line. Both NAD+ and IMP are bound (D). (E and F) Cryo-EM map of the S160del tetramer (E). Only the catalytic domain is resolved (green). The disordered Bateman domain is represented as a purple dashed line. Both NAD+ and IMP are bound (F).
Fig. 5. Ligand Binding restores polymerization, but not GTP regulation, to S160del. (A) Experimental diagram. S160del IMPDH2 tetramers are active, and the regulatory Bateman domain (purple) is highly flexible. Ap5G and GTP bind to the Bateman domains of tetramers and stabilize the formation of canonical octamers and filaments. (B) Negative stain electron micrographs of WT and S160del hIMPDH2 incubated with Ap5G and in the assay conditions from panel C. (C) In vitro enzyme assay of WT and S160del hIMPDH2 in the absence of any allosteric effectors (Left), in the presence of Ap5G (Middle), and in the presence of Ap5G and GTP (Right). Plotting mean of three technical replicates. Error bars represent SD.
