Abstract
Aspartate transcarbamoylase (ATCase) from Escherichia coli catalyzes a key step in pyrimidine nucleotide biosynthesis and has long served as a model for allosteric regulation. Despite decades of study, how nucleotide binding at distant regulatory sites controls cooperativity between active sites remained unresolved. Here we show that ATCase does not simply interconvert between two conformations, as traditionally depicted, but instead samples a continuum of conformations that tune enzyme cooperativity. Using complementary cryo-electron microscopy, small-angle X-ray scattering, and crystallography under conditions that ensure full assembly of the allosteric sites, we show that ATCase behaves like a flexible balloon whose global “breathing” motions directly regulate activity: compression enforces high cooperativity, inhibiting the enzyme, whereas expansion relieves this cooperativity and activates the enzyme. We further show that all four ribonucleoside triphosphates act in symmetric pairs to tune this motion, with the pyrimidines CTP and UTP compressing the enzyme to limit further pyrimidine production, and the purines ATP and GTP expanding it to balance pyrimidine and purine pools. Together, these findings uncover a dynamic breathing mechanism for long-range allosteric communication in ATCase.
Similar content being viewed by others
Data availability
Coordinates and structure factors for crystal structures have been deposited in the Protein Data Bank with accession codes: 9EEH (R: ATP/GTP/PALA) and 9EEJ (R: ATP/CP/SIN). Cryo-EM maps have been deposited to the Electron Microscopy Data Bank with accession codes: EMD-47956 (T), EMD-47957 (T: CTP/UTP/CP), EMD-47958 (R: CTP/UTP/CP/SIN), EMD-47959 (T: CTP/CP), EMD-47960 (R: CTP/CP/SIN), EMD-47961 (R: CP/SIN), EMD-47963 (R: ATP/CP/SIN), EMD-47964 (R: ATP/GTP/CP/SIN), EMD-47965 (R: ATP/GTP/CP), EMD-47966 (T: ATP/CP). The associated atomic models have been deposited to the Protein Data Bank with accession codes: 9EEK (T), 9EEL (T: CTP/UTP/CP), 9EEM (R: CTP/UTP/CP/SIN), 9EEN (T: CTP/CP), 9EEO (R: CTP/CP/SIN), 9EEP (R: CP/SIN), 9EEQ (R: ATP/CP/SIN), 9EER (R: ATP/GTP/CP/SIN), 9EES (R: ATP/GTP/CP), 9EEU (T: ATP/CP). SEC-SAXS datasets are available on Zenodo with record numbers: 17684630 (T), 17684632 (R), 17684634 (R: CTP), 17684636 (R: CTP/UTP), 17684638 (R: ATP), 17684640 (R: ATP/GTP). Source data are provided as a Source Data file. Source data are provided with this paper.
References
Gerhart, J. From feedback inhibition to allostery: the enduring example of aspartate transcarbamoylase. FEBS J. 281, 612–620 (2014).
Pardee, A. B. & Yates, R. A. Pyrimidine biosynthesis in Escherichia coli. J. Biol. Chem. 221, 743–756 (1956).
Pardee, A. B. & Yates, R. A. Control of pyrimidine biosynthesis in Escherichia coli by a feed-back mechanism. J. Biol. Chem. 221, 757–770 (1956).
Gerhart, J. C. & Pardee, A. B. The enzymology of control by feedback inhibition. J. Biol. Chem. 237, 891–896 (1962).
Monod, J., Wyman, J. & Changeux, J. P. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88–118 (1965).
Wiley, D. C. et al. The 5.5 Angstrom resolution structure of the regulatory enzyme, asparate transcarbamylase. Cold Spring Harb. Symp. Quant. Biol. 36, 285–290 (1972).
Krause, K. L., Volz, K. W. & Lipscomb, W. N. Structure at 2.9-A resolution of aspartate carbamoyltransferase complexed with the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Proc. Natl. Acad. Sci. USA 82, 1643–1647 (1985).
Ke, H., Lipscomb, W. N., Cho, Y. & Honzatko, R. B. Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase. J. Mol. Biol. 204, 725–747 (1988).
Jin, L., Stec, B., Lipscomb, W. N. & Kantrowitz, E. R. Insights into the mechanisms of catalysis and heterotropic regulation of Escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate at 2.1 A. Proteins 37, 729–742 (1999).
Honzatko, R. B. et al. Crystal and molecular structures of native and CTP-liganded aspartate carbamoyltransferase from Escherichia coli. J. Mol. Biol. 160, 219–263 (1982).
Kim, K. H., Pan, Z. X., Honzatko, R. B., Ke, H. M. & Lipscomb, W. N. Structural asymmetry in the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli. J. Mol. Biol. 196, 853–875 (1987).
Gouaux, J. E. & Lipscomb, W. N. Three-dimensional structure of carbamoyl phosphate and succinate bound to aspartate carbamoyltransferase. Proc. Natl. Acad. Sci. USA 85, 4205–4208 (1988).
Gouaux, J. E. & Lipscomb, W. N. Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-.ANG. resolution and neutral pH. Biochemistry 29, 389–402 (1990).
Gouaux, J. E., Stevens, R. C. & Lipscomb, W. N. Crystal structures of aspartate carbamoyltransferase ligated with phosphonoacetamide, malonate, and CTP or ATP at 2.8-.ANG. resolution and neutral pH. Biochemistry 29, 7702–7715 (1990).
Stevens, R. C., Gouaux, J. E. & Lipscomb, W. N. Structural consequences of effector binding to the T state of aspartate carbamoyltransferase: crystal structures of the unligated and ATP- and CTP-complexed enzymes at 2.6-Å resolution. Biochemistry 29, 7691–7701 (1990).
Stevens, R. C. & Lipscomb, W. N. A molecular mechanism for pyrimidine and purine nucleotide control of aspartate transcarbamoylase. Proc. Natl. Acad. Sci. USA 89, 5281–5285 (1992).
Peterson, A. W., Cockrell, G. M. & Kantrowitz, E. R. A second allosteric site in Escherichia coli aspartate transcarbamoylase. Biochemistry 51, 4776–4778 (2012).
Cockrell, G. M. & Kantrowitz, E. R. Metal ion involvement in the allosteric mechanism of Escherichia coli aspartate transcarbamoylase. Biochemistry 51, 7128–7137 (2012).
Cockrell, G. M. et al. New paradigm for allosteric regulation of Escherichia coli aspartate transcarbamoylase. Biochemistry 52, 8036–8047 (2013).
Velyvis, A., Yang, Y. R., Schachman, H. K. & Kay, L. E. A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase. Proc. Natl. Acad. Sci. USA 104, 8815–8820 (2007).
Velyvis, A., Schachman, H. K. & Kay, L. E. Application of methyl-TROSY NMR to test allosteric models describing effects of nucleotide binding to aspartate transcarbamoylase. J. Mol. Biol. 387, 540–547 (2009).
Tauc, P., Leconte, C., Kerbiriou, D., Thiry, L. & Hervé, G. Coupling of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase. J. Mol. Biol. 155, 155–168 (1982).
Hervé, G., Moody, M. F., Tauc, P., Vachette, P. & Jones, P. T. Quaternary structure changes in aspartate transcarbamylase studied by X-ray solution scattering. Signal transmission following effector binding. J. Mol. Biol. 185, 189–199 (1985).
Fetler, L., Tauc, P., Hervé, G., Moody, M. F. & Vachette, P. X-ray scattering titration of the quaternary structure transition of aspartate transcarbamylase with a bisubstrate analogue: influence of nucleotide effectors. J. Mol. Biol. 251, 243–255 (1995).
Fetler, L. & Vachette, P. The allosteric activator Mg-ATP modifies the quaternary structure of the R-state of Escherichia coli aspartate transcarbamylase without altering the T↔R equilibrium. J. Mol. Biol. 309, 817–832 (2001).
Svergun, D. I., Barberato, C., Koch, M. H., Fetler, L. & Vachette, P. Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state. Proteins 27, 110–117 (1997).
Wild, J. R., Loughrey-Chen, S. J. & Corder, T. S. In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase. Proc. Natl. Acad. Sci. USA 86, 46–50 (1989).
Moody, M. F., Vachette, P. & Foote, A. M. Changes in the X-ray solution scattering of aspartate transcarbamylase following the allosteric transition. J. Mol. Biol. 133, 517–532 (1979).
Tsuruta, H. et al. Kinetics of the quaternary structure change of aspartate transcarbamylase triggered by succinate, a competitive inhibitor. Biochemistry 33, 10007–10012 (1994).
Fetler, L., Kantrowitz, E. R. & Vachette, P. Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase. Proc. Natl. Acad. Sci. USA 104, 495–500 (2007).
Pastra-Landis, S. C., Evans, D. R. & Lipscomb, W. N. The effect of pH on the cooperative behavior of aspartate transcarbamylase from Escherichia coli. J. Biol. Chem. 253, 4624–4630 (1978).
Kosman, R. P., Gouaux, J. E. & Lipscomb, W. N. Crystal structure of CTP-ligated T state aspartate transcarbamoylase at 2.5 Å resolution: Implications for ATCase mutants and the mechanism of negative cooperativity. Proteins 15, 147–176 (1993).
West, J. M. et al. Time evolution of the quaternary structure of Escherichia coli aspartate transcarbamoylase upon reaction with the natural substrates and a slow, tight-binding inhibitor. J. Mol. Biol. 384, 206–218 (2008).
Meisburger, S. P., Xu, D. & Ando, N. REGALS: a general method to deconvolve X-ray scattering data from evolving mixtures. IUCrJ 8, 225–237 (2021).
Punjani, A. & Fleet, D. J. 3D variability analysis: resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM. J. Struct. Biol. 213, 107702 (2021).
Bennett, B. D. et al. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 5, 593–599 (2009).
Buckstein, M. H., He, J. & Rubin, H. Characterization of nucleotide pools as a function of physiological state in Escherichia coli. J. Bacteriol. 190, 718–726 (2008).
Patterson, M. G. Revisiting Allostery in Escherichia coli Aspartate Transcarbamoylase. (search.proquest.com, 2024).
Chen, H., Giese, T. J., Golden, B. L. & York, D. M. Divalent metal ion activation of a guanine general base in the hammerhead ribozyme: insights from molecular simulations. Biochemistry 56, 2985–2994 (2017).
Purcarea, C., Hervé, G., Ladjimi, M. M. & Cunin, R. Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli. J. Bacteriol. 179, 4143–4157 (1997).
Sakash, J. B. & Kantrowitz, E. R. The N-terminus of the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for both nucleotide binding and heterotropic effects. Biochemistry 37, 281–288 (1998).
Dembowski, N. J. & Kantrowitz, E. R. The use of alanine scanning mutagenesis to determine the role of the N-terminus of the regulatory chain in the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase. Protein Eng. Des. Sel. 7, 673–679 (1994).
Kenny, M. J., McPhail, D. & Shepherdson, M. A reappraisal of the diversity and class distribution of aspartate transcarbamoylases in gram-negative bacteria. Microbiology 142, 1873–1879 (1996).
Stebbins, J. W. & Kantrowitz, E. R. Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution. Biochemistry 31, 2328–2332 (1992).
Pastra-Landis, S. C., Foote, J. & Kantrowitz, E. R. An improved calorimetric assay for aspartate and ornithine transcarbamylases. Anal. Biochem. 118, 358–363 (1981).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Kimanius, D., Dong, L., Sharov, G., Nakane, T. & Scheres, S. H. W. New tools for automated cryo-EM single-particle analysis in RELION-4.0. Biochem. J. 478, 4169–4185 (2021).
Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).
Winter, G. et al. DIALS as a toolkit. Protein Sci. 31, 232–250 (2022).
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Acknowledgements
The authors thank S. Meisburger, J. Bacik, D. Xu, J. Kaelber, and the Phenix team for technical discussions and B. Barstow, H. Wang, J. Dalal, and R. Schweinfurth for critical reading of this manuscript. We are grateful to the staff and resources at CHESS, CCMR, APS, NCCAT, and the Cornell NMR and Chemistry Mass Spectrometry Facilities. We thank C. Aplin, M. Lynch, and R. Schweinfurth for assistance with data collection. SAXS/crystallography were conducted at the Center for High-Energy X-ray Sciences (CHEXS), which is supported by the National Science Foundation award DMR-2342336, and the MacCHESS facility, which is supported by award 1-P30-GM124166 from the National Institute of General Medical Sciences (NIGMS) and the National Institutes of Health (NIH). Cryo-crystallography was conducted at NE-CAT, which is funded by the NIGMS from the NIH (P30 GM124165), using resources of APS, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under DE-AC02-06CH11357. Cryo-EM performed at NCCAT was supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program grant U24 GM129539. This work was supported by a half-time student appointment at CHEXS (to R.C.M.), Cornell Provost Diversity Fellowship (to M.G.P.), NIH grant GM124847 (to N.A.), and startup funds from Cornell University (to N.A.).
Author information
Authors and Affiliations
Contributions
R.C.M. and M.G.P. purified proteins and performed SAXS experiments. R.C.M. performed SAXS data analysis, kinetic modeling of assay data, cryo-EM experiments, and cryo-EM data processing, model building, and refinement. M.G.P. performed activity assays, kinetic modeling, and crystallographic and cryo-EM model building and refinement. M.G.P. and N.B. grew crystals and performed crystallography experiments. X.P. synthesized PALA. N.A. supervised the project, acquired funding, and performed data analysis. R.C.M., M.G.P., and N.A. made figures and wrote the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks Jan Felix, George P. Lisi, Paul Sauer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Miller, R.C., Patterson, M.G., Bhatt, N. et al. Cooperativity in E. coli aspartate transcarbamoylase is tuned by allosteric breathing. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70909-y
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-70909-y
