Abstract
Inosine-5′-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.
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Data availability
The coordinates are deposited in the PDB with accession codes PDB 7RER (interface-centered extended IMPDH1(514)), PDB 7RES (octamer-centered active IMPDH1(514)), PDB 7RFE (interface-centered compressed IMPDH1(514)), PDB 7RFG (octamer-centered compressed IMPDH1(514)), PDB 7RGL (interface-centered extended IMPDH1(546)), PDB 7RGM (octamer-centered extended IMPDH1(546)), PDB 7RGI (interface-centered compressed IMPDH1(546)), PDB 7RGQ (octamer-centered compressed IMPDH1(546)), PDB 7RFF (interface-centered extended IMPDH1(595)), PDB 7RFH (octamer-centered extended IMPDH1(595)), PDB 7RFI (interface-centered compressed IMPDH1(595)), PDB 7RGD (octamer-centered compressed IMPDH1(595). The cryo-EM maps are deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-24437 (interface-centered extended IMPDH1(514)), EMD-24438 (octamer-centered extended IMPDH1(514)), EMD-24439 (interface-centered compressed IMPDH1(514)), EMD-24441 (octamer-centered compressed IMPDH1(514)), EMD-24451 (interface-centered extended IMPDH1(546)), EMD-24452 (octamer-centered extended IMPDH1(546)), EMD-24450 (interface-centered compressed IMPDH1(546)), EMD-24454 (octamer-centered compressed IMPDH1(546)), EMD-24440 (interface-centered extended IMPDH1(595)), EMD-24442 (octamer-centered extended IMPDH1(595)), EMD-24443 (interface-centered compressed IMPDH1(595)) and EMD-24448 (octamer-centered compressed IMPDH1(595). Source data are provided with this paper.
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Acknowledgements
We thank the Arnold and Mabel Beckman Cryo-EM Center at the University of Washington for electron microscope use. We thank J. Calise for valuable feedback on the manuscript. This work was supported by the US National Institutes of Health (grant nos. R01GM118396 and R21EY031546 and S10OD032290 to J.M.K., R01GM-083025 to J.R.P., T32 CA-009035 to J.C.S. and F31EY030732 and T32GM008268 to A.L.B.), Universidad de Salamanca (Junta de Castilla y León fellowship to D.F.-J.), and Ministerio de Economía y Competitividad (grant no. BFU2016-79237-P to R.M.B.).
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A.L.B. performed kinetics assays, protein purification, cryo-EM data collection and image processing, and structure analysis. C.N. optimized kinetics experiment design. M.S. and M.C.J. developed cryo-EM processing approaches and processed cryo-EM data. J.C.S. designed and performed immunofluorescence experiments. D.F.-L. produced WT retinal protein used for cryo-EM. J.Q. optimized cryo-EM sample preparation. R.M.B. and J.R.P. conceptualized experiments. A.L.B. and J.M.K designed experiments, performed data analysis and interpretation and wrote the manuscript.
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Extended data
Extended Data Fig. 1 IMPDH structure and function.
a, Purine biosynthesis pathway. b, IMPDH monomer (6u9o) has a catalytic domain (green) that binds IMP and NAD+ in the active site, and a regulatory domain (pink) with three allosteric nucleotide binding sites. c, IMPDH is a tetramer in solution and can adopt a flat or bowed conformation. Side view of tetramers are depicted, so that only two monomers are visible. d, ATP (sites 1&2) or GTP (sites 2&3) binding promotes octamer assembly. e, IMPDH2 octamers can assemble into filaments of stacked octamers.
Extended Data Fig. 2 IMPDH1 Sequence Alignment.
Evolutionary conservation of the helix in the N-terminus of the longer retinal splice variant (blue) and the first 12 canonical residues particularly tyrosine 12 (pink).
Extended Data Fig. 3 IMPDH2-WT filament resists GTP inhibition.
a,b, GTP inhibition curves of IMPDH2 or IMPDH1-WT (solid line) and the respective non-assembly Y12A protein (dashed line). Individual data points are shown as diamonds (IMPDH2) or circles (IMPDH1), where filled are WT and empty Y12A. Reactions were performed in triplicate and the average for each concentration is shown as a bold rectangle (filled is WT, empty is Y12A). Error bars are standard deviation calculated from n = 3. Reactions performed with 1 µM protein, 1 mM ATP, 1 mM IMP, 300 µM NAD+, and varying GTP.
Extended Data Fig. 4 Cryo-EM workflow.
Flow chart summarizing data processing strategy for IMPDH1+ ATP/IMP/NAD+.
Extended Data Fig. 5 IMPDH1 active site map and model.
a-f, Cartoon representation of the active site. Side chains around the active site are shown as sticks. Chain A is dark green while the neighboring chain is light green. NAD+ is yellow and IMP red. Density for the ligand(s) is shown as a surface. a, IMPDH1(514) bound to ATP/IMP/NAD+. b, IMPDH1(514) bound to GTP/ATP/IMP. c, IMPDH1(546) bound to ATP/IMP/NAD+. d, IMPDH1(546) bound to GTP/ATP/IMP/NAD+. e, IMPDH1(595) bound to ATP. f, IMPDH1(595) bound to GTP/ATP/IMP/NAD+.
Extended Data Fig. 6 Inhibited IMPDH1-WT tetramer is in a bowed conformation.
a, Comparison of the catalytic tetramers of inhibited IMPDH2 filament (gray; 6u8s) to inhibited IMPDH2 free octamer (6uaj). Aligned on monomers with asterisk, other monomer pair has an alpha carbon RMSD of 2.1 Å. b, Comparison of the catalytic tetramers of inhibited IMPDH2 filament (gray; 6u8s) to inhibited IMPDH1 filament. Aligned on monomers with asterisk, other monomer pair has an alpha carbon RMSD of 3.7 Å.
Extended Data Fig. 7 Y12A non-assembly mutations prevents assembly in IMPDH1 variants.
Negative stain EM of purified human IMPDH1. Non-assembly mutation Y12A breaks both ATP- and GTP-dependent assembly. Scale bar 100 nm. Reactions performed with 1 µM protein, 1 mM ATP if used, 1 mM GTP if used.
Extended Data Fig. 8 IMPDH1 retinal variant (546) is similar to canonical IMPDH1.
a-c, Active IMPDH1(546) filament bound to ATP, IMP, NAD+. a, Low-pass filtered cryo-EM reconstruction b, Interface-focused cryo-EM reconstruction. 8 monomers are colored by catalytic domain (green) and regulatory domain (pink). c, View of the top of an octamer from inside the filament. The surface area buried by the octamer interface is in aqua with the indicated total buried surface area. (Surface representation of the atomic model at the assembly interface, with buried residues in cyan). d-f, Inhibited IMPDH1(546) filament bound to GTP, ATP, IMP, NAD+. d, Low-pass filtered cryo-EM reconstruction e, Interface-focused cryo-EM reconstruction. 8 monomers are colored by catalytic domain (green) and regulatory domain (pink). f, View of the top of an octamer from inside the filament. The surface area buried by the octamer interface is in aqua with the indicated total buried surface area. (Surface representation of the atomic model at the assembly interface, with buried residues in cyan).
Extended Data Fig. 9 IMPDH1 Retinal Variant C-term disrupts interactions.
a, Evolutionary conservation of the C-terminus in canonical IMPDH1 and both retinal splice variants. b, Surface representation of octamer side view. Dotted box indicates the region shown in c-d. c-d, Each chain is a different color green, C-term residues 510–512 in orange, and IMP in purple. c, inhibited canonical IMPDH1. d, Inhibited retinal variant IMPDH1(546).
Extended Data Fig. 10 IMPDH1 disease mutants have a variety of assembly phenotypes.
Negative stain EM of purified human IMPDH1. Scale bar 100 nm. Reactions performed with 1 µM protein, 1 mM ATP, 5 mM GTP, 3 mM IMP, 5 mM NAD+.
Supplementary information
Supplementary Information
Supplementary Tables 1–4.
Supplementary Video 1.
Comparison between large and small interface IMPDH1 filaments. Morph comparison between large interface of ATP/IMP/NAD+ IMPDH1 filament to small interface in GTP/ATP/IMP IMPDH1 filament.
Supplementary Data 1
Supplementary data for Supplemental Table 1.
Supplementary Data 2
Supplementary data for Supplemental Table 4.
Source data
Source Data Fig. 1
Statistical source data.
Source Data Fig. 4
Statistical source data.
Source Data Fig. 6
Statistical source data.
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Burrell, A.L., Nie, C., Said, M. et al. IMPDH1 retinal variants control filament architecture to tune allosteric regulation. Nat Struct Mol Biol 29, 47–58 (2022). https://doi.org/10.1038/s41594-021-00706-2
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DOI: https://doi.org/10.1038/s41594-021-00706-2
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