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Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

Nature Structural & Molecular Biology volume 27, pages 42–48 (2020)Cite this article

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Abstract

Many enzymes assemble into defined oligomers, providing a mechanism for cooperatively regulating activity. Recent studies have described a mode of regulation in which enzyme activity is modulated by polymerization into large-scale filaments. Here we describe an ultrasensitive form of polymerization-based regulation employed by human CTP synthase 2 (CTPS2). Cryo-EM structures reveal that CTPS2 filaments dynamically switch between active and inactive forms in response to changes in substrate and product levels. Linking the conformational state of many CTPS2 subunits in a filament results in highly cooperative regulation, greatly exceeding the limits of cooperativity for the CTPS2 tetramer alone. The structures reveal a link between conformation and control of ammonia channeling between the enzyme’s active sites, and explain differences in regulation of human CTPS isoforms. This filament-based mechanism of enhanced cooperativity demonstrates how the widespread phenomenon of enzyme polymerization can be adapted to achieve different regulatory outcomes.

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Fig. 1: CTPS2 forms distinct S- and P-state filaments using the same filament interface.
Fig. 2: Product binding and changes in the tetramer interface in CTPS2 structures.
Fig. 3: UTP binding opens a tunnel connecting the amidoligase and glutaminase active sites in the S-state CTPS2 filament.
Fig. 4: Disulfide-cross-linked CTPS2 filaments can switch conformations.
Fig. 5: Regulation of CTPS2 filaments is highly cooperative.

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Data availability

Cryo-EM structures and atomic models have been deposited in the EMDB and PDB, respectively, with accession codes EMD-20354, PDB 6PK4 (S-state CTPS2 filament) and EMD-20355, PDB 6PK7 (P-state CTPS2 filament).

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Acknowledgements

We thank G. Carman (Rutgers University) for the CTPS-expressing S. cerevisiae strains. We thank E. Baldwin (UC Davis), who provided valuable feedback on our experimental results and early drafts of this manuscript. We are grateful to the Arnold and Mabel Beckman Cryo-EM Center at the University of Washington for use of electron microscopes. This work was supported by the US National Institutes of Health (grant no. R01 GM118396 to J.M.K.).

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Authors and Affiliations

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

    Eric M. Lynch & Justin M. Kollman

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Contributions

E.M.L. performed the experiments. E.M.L. and J.M.K. designed experiments, performed data analysis and interpretation and wrote the manuscript.

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Correspondence to Justin M. Kollman.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 CryoEM data processing flowchart for the S-state CTPS2 filament.

Additional details are provided in the Methods section.

Extended Data Fig. 2 CryoEM data processing flowchart for the P-state CTPS2 filament.

Additional details are provided in the Methods section.

Extended Data Fig. 3 Details of CTPS2 cryoEM structures.

a,b FSC curves for the S-state (a) and P-state (b) CTPS2 filament structures, showing resolutions of 3.5Å and 3.1Å, respectively, by the FSC0.143 criteria. c,d, ResMap local resolution maps of the S-state (c) and P-state (d) CTPS2 filament structures. e, CTPS2 tetramer, with monomers A-D shown in different colors. f, Zoomed-in view of the orange box in (e), showing S-state CTPS2 (blue), P-state CTPS2 (dark grey), and S-state CTPS1 (light grey) filament structures aligned on the amidoligase domain of subunit A. S-state CTPS1 and CTPS2 are extended across the tetramer interface, compared to P-state CTPS2. g, Monomers from the S-state (green) and P-state (dark grey) CTPS2 filament structures aligned on the amidoligase domain. h, Zoomed-in view of the blue box in (g), with S-State CTPS1 also shown in light grey. The glutaminase domain is rotated by 7o towards the amidoligase domain in the S-state structure. i,j Two views of the S-state (color) and P-state (grey) CTPS2 monomers aligned on the glutaminase-amidoligase interface. k, Zoomed-in view of the yellow box in (i). l, Zoomed-in view of the orange box in (j). The glutaminase-amidoligase interface is essentially identical (Cα RMSD 0.8 Å) in the S-state and P-state filaments.

Extended Data Fig. 4 Rare apo CTPS2 filaments have S-state filament architecture.

a, Negative stain EM images of apo CTPS2. Occasional filaments are observed. b, Helical rise (red) and rotation (black) values plotted over multiple rounds of iterative helical real space reconstruction of apo CTPS2 filaments in stain. Starting helical symmetry values and models from cryoEM structures of S-state (closed circles) or P-state (open circles) CTPS2 filaments were used. Both reconstructions converge on the S-state filament helical symmetry values. c, The apo CTPS2 filament structures from the reconstructions described in (b) are the same and have the S-state helical rotation, regardless of which starting symmetries and models are used. Scale bars are 50 nm.

Extended Data Fig. 5 Product-bound CTPS1 tetramers occasionally associate.

a, Negative stain EM images of CTPS1 in the presence of CTP and ADP. b, Zoomed-in view of the blue box in (a). The majority of CTPS1 is tetrameric, but rare pairs of tetramers are also observed (orange box). c, Reference-free 2D averages of CTPS1 in the presence of CTP and ADP. The percentage of all particles in each class is indicated. A single class containing pairs of tetramers (orange box) accounts for 3% of all particles. d, Negative stain EM reconstruction of the product-bound CTPS1 tetramer at 18Å resolution. The overall architecture resembles structures of existing CTPS tetramers. A CTPS1 tetramer from PBD 5U03 is fit for comparison. Scale bars are 50 nm.

Extended Data Fig. 6 The CTPS1 C-terminus is required for filament formation.

a, CryoEM maps of CTPS1 and CTPS2 filaments, low-pass filtered to 8Å for comparison. CTPS1 filaments have an additional C-terminal filament contact (orange) that is not observed in S- or P-state CTPS2 filaments. In the CTPS2 filament structures, the distance between adjacent C-termini increases by 8Å upon transition from the S-state to the P-state conformation. b, Sequence alignment of the CTPS1 and CTPS2 C-termini. Conserved residues are shown in blue. The C-termini have 41% sequence identity, compared with 75% overall sequence identity. c, Negative stain EM images of CTPS2-∆C, wild-type CTPS1, and CTPS1-∆C in the presence of substrates or products. CTPS1-∆C does not form filaments. d, Model for the role of the C-terminus in CTPS1 and CTPS2 polymerization. The CTPS1 C-terminus forms a contact that stabilizes the S-state filament, but is incompatible with the P-state conformation. The CTPS2 C-terminus is not required for polymerization. The lack of density for the CTPS2 C-terminus in the S- and P-state structures suggests it is disordered. e, Sequence alignment of CTPS1 and CTPS2 primary filament contacts, with differing residues highlighted in red. f,g CTPS1 (f) and CTPS2 (g) primary filament contact. Residues 347 and 354 adjacent to conserved H355, which differ between the two structures, are shown in orange and yellow. Scale bars are 50 nm.

Extended Data Fig. 7 CryoEM density at the P52-V58 and H55 constriction points.

a,b CryoEM density (grey) and atomic models (color) at the H55 gate in the P-state (a) and S-state (b) CTPS2 filaments. c,d CryoEM density (grey) and atomic models (color) at the P52-V58 constriction in the P-state (c) and S-state (d) CTPS2 filaments.

Extended Data Fig. 8 CTPS2cc forms disulfide crosslinks.

SDS-PAGE gel of wild-type CTPS2 and CTPS2CC under reducing (100 mM DTT) and non-reducing conditions. Bands for crosslinked CTPS2CC are visible under non-reducing conditions.

Extended Data Fig. 9 ADP-Glo assays show a linear response in relative light units (RLU) over the time used for CTPS2 kinetics assays.

a, Time-course of ADP-Glo assay under the substrate conditions used for CTP inhibition assays at 300 nM CTPS2 (open circles) and 1500 nM CTPS2 (closed circles). Rates were calculated from 12 minute timepoints for 1500 nM CTPS2 and 60 minutes for 300 nM CTPS2. b, Time-course of ADP-Glo assay at 1500 nM CTPS2 with the lowest UTP concentration used in UTP kinetics assays (10 µM UTP). Dashed lines indicate the linear portion of the assay used to measure reaction velocity. Data shown are mean and s.d. of n = 3 technical replicates for all panels.

Supplementary information

Supplementary Information

Supplementary Table 1.

Reporting Summary

Supplementary Video 1

Animation of the CTPS2 active site morphing between the S-state and P-state conformations. The glutaminase domain (green) rotates towards the amidoligase domain (blue), while loop P52–V58 (orange) is pulled towards the UTP base (magenta) in the S-state. ATP (yellow) is also shown in the active site.

Supplementary Video 2

Animation of CTPS2 morphing between the S-state and P-state filament conformations. Colored by tetramer. Filament contact sites (orange) remain the same despite conformational changes between the two filament states.

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Lynch, E.M., Kollman, J.M. Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments. Nat Struct Mol Biol 27, 42–48 (2020). https://doi.org/10.1038/s41594-019-0352-5

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