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Human CTP synthase filament structure reveals the active enzyme conformation

Nature Structural & Molecular Biology volume 24, pages 507–514 (2017)Cite this article

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Abstract

The universally conserved enzyme CTP synthase (CTPS) forms filaments in bacteria and eukaryotes. In bacteria, polymerization inhibits CTPS activity and is required for nucleotide homeostasis. Here we show that for human CTPS, polymerization increases catalytic activity. The cryo-EM structures of bacterial and human CTPS filaments differ considerably in overall architecture and in the conformation of the CTPS protomer, explaining the divergent consequences of polymerization on activity. The structure of human CTPS filament, the first structure of the full-length human enzyme, reveals a novel active conformation. The filament structures elucidate allosteric mechanisms of assembly and regulation that rely on a conserved conformational equilibrium. The findings may provide a mechanism for increasing human CTPS activity in response to metabolic state and challenge the assumption that metabolic filaments are generally storage forms of inactive enzymes. Allosteric regulation of CTPS polymerization by ligands likely represents a fundamental mechanism underlying assembly of other metabolic filaments.

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Figure 1: Mechanism of ecCTPS assembly.
Figure 2: Engineered disulfides drive ecCTPS assembly and inhibit activity.
Figure 3: hCTPS1 filaments assemble with substrates and are catalytically active.
Figure 4: hCTPS1 filaments reveal the active conformation of CTP synthase.
Figure 5: ecCTPS and hCTPS1 undergo a conserved conformational cycle controlled by substrate and product binding.

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Acknowledgements

The authors are grateful to G. Carman (Rutgers University) for the hCTPS1-expressing S. cerevisiae strain. This work was supported by the US National Institutes of Health (R01 GM118396 to J.M.K.) and the Human Frontier Science Program (RGY0076/2013 to Z.G. and J.M.K.). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

Author information

Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, Washington, USA

    Eric M Lynch, Derrick R Hicks, Allison Maker, Jesse M Hansen & Justin M Kollman

  2. Graduate Program in Molecular and Cellular Biology, University of Washington, Seattle, Washington, USA

    Derrick R Hicks

  3. Department of Biology, McGill University, Montreal, Quebec, Canada

    Matthew Shepherd

  4. Department of Molecular and Cellular Biology, University of California, Davis, Davis, California, USA.,

    James A Endrizzi & Enoch P Baldwin

  5. Graduate Program in Biological Physics, Structure, and Design, University of Washington, Seattle, Washington, USA

    Jesse M Hansen

  6. Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA

    Rachael M Barry & Zemer Gitai

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Contributions

E.M.L. and J.M.K. wrote the manuscript. E.M.L., J.M.K., and D.R.H. solved the EM structures. J.A.E. and E.P.B. solved the crystal structure of ecCTPS. M.S. and J.M.H. generated disulfide cross-linked ecCTPS constructs and optimized purification and assembly conditions. E.M.L. and A.M. carried out CTPS activity assays. R.M.B. and Z.G. performed initial characterization of ecCTPS filaments. E.M.L., Z.G., E.P.B. and J.M.K. contributed to experimental design and data analysis and interpretation.

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Correspondence to Justin M Kollman.

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Integrated supplementary information

Supplementary Figure 1 Cryo-EM reconstruction of ecCTPS.

a, Cryo-electron micrograph of ecCTPS filaments. b, The resolution of the structure is 4.6 Å by gold-standard FSC. c, Local resolution map of one ecCTPS tetramer in the filament. d, A central slice through the unfiltered ecCTPS filament reconstruction shows less well-defined features in the GAT domains (yellow arrow) than in the AL domains (white arrowhead).

Supplementary Figure 2 Details of structural rearrangements at the CTP binding site that create new intersubunit contacts and drive the conformational switch to the filament-forming conformer.

a, The alternative ecCTPS-CTP complex structure (5TKV) is shown in yellow (subunit A), red (subunit B) and green (subunit A’, two-fold symmetry related to A, symmop 4). The previously published CTP complex (2AD5)16 is shown in gray, after superposition of the A subunits (residues 1-266, 0.42 Å Cα rmsd after superposition). Side chains that have altered interactions or conformations relative to 2AD5 are displayed. Hydrogen bonds unique to 5TKV are shown as cyan dotted lines, and heavy atom distances are indicated. The 2AD5 CTP is depicted using narrow sticks. In 5TKV, an octahedral Mg+2 ion coordinates the β- and γ-phosphate oxygens and 3 water molecules. Relative to the A subunit, helices α5(A’) and α7(A) Cα atoms shifted 1.9 Å and 1.1 Å respectively. F227 and N229 side chains changed rotamers, with the F227 phenyl ring packed against the CTP C5 atom, while N229 formed new inter-subunit hydrogen bonds with R158. These new hydrogen bonds became possible due to a quaternary rearrangement of subunits that was similar to the rearrangement of N-terminal domains in the filament-binding conformer observed by cryoEM. This structure validates the sequence conservation for these residues. Mutations at E155, R158 and position 229 confer pyrimidine drug resistance in CTPSs, presumably by hindering the conformational change required for formation of the filament (bacterial49) or inhibited tetramer (yeast50, mammalian51). Similar intersubunit contacts are observed in tetramer crystal structures of the M. tuberculosis CTPS-diUTP complex (PDB 4ZDJ), T. thermophilus CTPS-glutamine (PDB 1VCO) and apo (PDB 1VCM) complexes, and the hCTPS1 AL domain (PDB 2VO1). b, E155 and N229 participate in extended hydrogen bond networks in the A’ and B subunits. Hydrogen bonds specific to 5TKV are depicted in magenta, while those common to both 2AD5 and 5TKV are colored light blue. R158 and N229 form the center of this network. 49. Wylie, J.L., Wang, L.L., Tipples, G. & McClarty, G. A single point mutation in CTP synthetase of Chlamydia trachomatis confers resistance to cyclopentenyl cytosine. J Biol Chem 271, 15393-400 (1996). 50. Ostrander, D.B., O'Brien, D.J., Gorman, J.A. & Carman, G.M. Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae. J Biol Chem 273, 18992-9001 (1998). 51. Whelan, J., Phear, G., Yamauchi, M. & Meuth, M. Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells. Nat Genet 3, 317-22 (1993).

Supplementary Figure 3 CTP promotes disassembly of hCTPS1 filaments.

a, Adding glutamine to hCTPS1 filaments assembled with UTP, ATP, and GTP causes them to disassemble over time. b, Adding glutamine to hCTPS1 filaments assembled with UTP, AMPPNP, and GTP does not lead to filament disassembly, suggesting CTP synthesis is required to promoted hCTPS1 depolymerization. c, Directly adding CTP to hCTPS1 filaments assembled with UTP, ATP, and GTP causes filaments to disassemble. Scale bars are 50 nm.

Supplementary Figure 4 Cryo-EM reconstruction of hCTPS1.

a, Section from a micrograph of frozen hCTPS1. b, FSC curve for the hCTPS1 filament, showing a resolution of 6.1Å. c, ResMap local resolution map of the hCTPS1 filament. d, Atomic model of the hCTPS1 monomer fit into the hCTPS1 filament cryoEM map, colored by domain. e, Individual helices are well-defined in the hCTPS1 filament cryoEM map. f, Comparison of the ecCTPS and hCTPS1 filament contact sites. hCTPS1 filaments lack the linker-linker contact site, and have a completely rearranged GAT contact site. g, Poorly-resolved C-terminal contact site (orange) in the hCTPS1 filament.

Supplementary Figure 5 Mutation of a conserved histidine in the eukaryotic GAT-domain insert abolishes hCTPS1 polymerization without disrupting tetramer formation.

a, CTPS sequence alignments, showing the appearance of the GAT domain helical insert early in eukaryotic evolution. The conserved histidine (H355 in humans) is shown in red. b, hCTPS1-H355A does not polymerize in the presence of UTP, ATP, and GTP. c, Negative stain reconstruction of the hCTPS1-H355A tetramer in the presence of substrates (grey), fit with an atomic model of hCTPS1 in the filament conformation (colored by protomer). Scale bar is 50 nm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5. (PDF 10328 kb)

Supplementary Video 1

Animation of ecCTPS tetramer morphing between the apo conformation and product-bound filament conformation. Monomers are shown in different colors, and filament contact sites are shown in red. (MOV 6109 kb)

Supplementary Video 2

Animation of ecCTPS tetramer morphing between the apo conformation and product-bound filament conformation (alternate view). Same animation as in Video 1, viewed down the filament axis. (MOV 3292 kb)

Supplementary Video 3

Animation of hCTPS1 tetramer morphing between the apo conformation and substrate-bound filament conformation. Monomers are shown in different colors, and filament contact sites are shown in red. (MOV 8709 kb)

Supplementary Video 4

Animation of hCTPS1 tetramer morphing between the apo conformation and substrate-bound filament conformation (alternate view). Same animation as in Video 3, viewed down the filament axis. (MOV 7319 kb)

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Lynch, E., Hicks, D., Shepherd, M. et al. Human CTP synthase filament structure reveals the active enzyme conformation. Nat Struct Mol Biol 24, 507–514 (2017). https://doi.org/10.1038/nsmb.3407

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