Significance

Conformational dynamics are likely critical to the activity of many multi-step enzymes. However, it is challenging to map the small-amplitude (sub-angstrom) and fast (nanosecond) dynamics that may underlie active site rearrangements. Here, the authors present X-ray diffraction experiments and analyses that allow for the sensitive identification of coupled motions in the enzyme dihydrofolate reductase. These methods resolve rapid structural changes throughout the enzyme that modify the active site and regulate access of water to the substrate, serving as an allosteric switch that organizes the enzyme’s two-step catalytic mechanism. The methods presented here can be used to characterize enzyme dynamics in unprecedented detail.

Abstract

Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident with Escherichia coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps.

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Data, Materials, and Software Availability

All structures determined in this study have been deposited in the Protein Databank with IDs: 8DAI (55), 5SSS (56), 5SST (57), 5SSU (58), 5SSV (59), 5SSW (60), 7FPL (61), 7FPM (62), 7FPN (63), 7FPO (64), 7FPP (65), 7FPQ (66), 7FPR (67), 7FPS (68), 7FPT (69), 7FPU (70), 7FPV (71), 7FPW (72), 7FPX (73), 7FPY (74), 7FPZ (75), 7FQ0 (76), 7FQ1 (77), 7FQ2 (78), 7FQ3 (79), 7FQ4 (80), 7FQ5 (81), 7FQ6 (82), 7FQ7 (83), 7FQ8 (84), 7FQ9 (85), 7FQA (86), 7FQB (87), 7FQC (88), 7FQD (89), 7FQE (90), 7FQF (91), 7FQG (92), 8G4Z (93), and 8G50 (94), as referenced in SI Appendix, Tables 1–11. Python and PyMOL scripts for generating figures, along with (difference) electron density maps are deposited in Zenodo (https://doi.org/10.5281/zenodo.7634123) (95). Crystallographic analyses make use of reciprocalspaceship and rs-booster, which are available from https://rs-station.github.io/ (96, 97). The forcefields, starting models, and scripts for reproducing the molecular dynamics trajectories are included in the Zenodo deposition.

Acknowledgments

We thank Drs. B. Correia, S. Eddy, R. Gaudet, and R. Losick for comments on the manuscript. D.R.H. is supported by the Searle Scholarship Program (SSP-2018-3240), a fellowship from the George W. Merck Fund of the New York Community Trust (338034), and the NIH Director’s New Innovator Award (DP2-GM141000). J.B.G. was supported by the NSF Graduate Research Fellowship under Grant No. DGE1745303. K.M.D. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. M.A.K. is supported by the NSF-Simons Center for Mathematical and Statistical Analysis of Biology at Harvard (award number #1764269) and the Harvard Quantitative Biology Initiative. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, National Institute of General Medical Sciences (including P41GM103393). In addition, this research used resources of the Advanced Photon Source, a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of BioCARS was also supported by the National Institute of General Medical Sciences of the NIH under grant number P41 GM118217. The time-resolved set-up at Sector 14 was funded in part through a collaboration with Philip Anfinrud (National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of National Institute of General Medical Sciences or NIH.

Author contributions

J.B.G., K.M.D., and D.R.H. designed research; J.B.G., K.M.D., D.E.B., M.A.K., C.J.S., I.-S.K., R.W.H., S.R., and D.R.H. performed research; J.B.G. contributed new reagents/analytic tools; J.B.G. and D.R.H. analyzed data; and J.B.G. and D.R.H. wrote the paper.

Competing interests

The authors declare no competing interest.

Supporting Information

Appendix 01 (PDF)

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Go to Proceedings of the National Academy of Sciences
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Proceedings of the National Academy of Sciences
Vol. 121 | No. 9
February 27, 2024
PubMed: 38386706

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Data, Materials, and Software Availability

All structures determined in this study have been deposited in the Protein Databank with IDs: 8DAI (55), 5SSS (56), 5SST (57), 5SSU (58), 5SSV (59), 5SSW (60), 7FPL (61), 7FPM (62), 7FPN (63), 7FPO (64), 7FPP (65), 7FPQ (66), 7FPR (67), 7FPS (68), 7FPT (69), 7FPU (70), 7FPV (71), 7FPW (72), 7FPX (73), 7FPY (74), 7FPZ (75), 7FQ0 (76), 7FQ1 (77), 7FQ2 (78), 7FQ3 (79), 7FQ4 (80), 7FQ5 (81), 7FQ6 (82), 7FQ7 (83), 7FQ8 (84), 7FQ9 (85), 7FQA (86), 7FQB (87), 7FQC (88), 7FQD (89), 7FQE (90), 7FQF (91), 7FQG (92), 8G4Z (93), and 8G50 (94), as referenced in SI Appendix, Tables 1–11. Python and PyMOL scripts for generating figures, along with (difference) electron density maps are deposited in Zenodo (https://doi.org/10.5281/zenodo.7634123) (95). Crystallographic analyses make use of reciprocalspaceship and rs-booster, which are available from https://rs-station.github.io/ (96, 97). The forcefields, starting models, and scripts for reproducing the molecular dynamics trajectories are included in the Zenodo deposition.

Submission history

Received: August 1, 2023
Accepted: December 18, 2023
Published online: February 22, 2024
Published in issue: February 27, 2024

Keywords

  1. protein dynamics
  2. allostery
  3. enzyme catalysis
  4. X-ray crystallography
  5. DHFR

Acknowledgments

We thank Drs. B. Correia, S. Eddy, R. Gaudet, and R. Losick for comments on the manuscript. D.R.H. is supported by the Searle Scholarship Program (SSP-2018-3240), a fellowship from the George W. Merck Fund of the New York Community Trust (338034), and the NIH Director’s New Innovator Award (DP2-GM141000). J.B.G. was supported by the NSF Graduate Research Fellowship under Grant No. DGE1745303. K.M.D. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. M.A.K. is supported by the NSF-Simons Center for Mathematical and Statistical Analysis of Biology at Harvard (award number #1764269) and the Harvard Quantitative Biology Initiative. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, National Institute of General Medical Sciences (including P41GM103393). In addition, this research used resources of the Advanced Photon Source, a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of BioCARS was also supported by the National Institute of General Medical Sciences of the NIH under grant number P41 GM118217. The time-resolved set-up at Sector 14 was funded in part through a collaboration with Philip Anfinrud (National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of National Institute of General Medical Sciences or NIH.
Author Contributions
J.B.G., K.M.D., and D.R.H. designed research; J.B.G., K.M.D., D.E.B., M.A.K., C.J.S., I.-S.K., R.W.H., S.R., and D.R.H. performed research; J.B.G. contributed new reagents/analytic tools; J.B.G. and D.R.H. analyzed data; and J.B.G. and D.R.H. wrote the paper.
Competing Interests
The authors declare no competing interest.

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Department of Molecular & Cellular Biology, Harvard University, Cambridge, MA 02138
Department of Molecular & Cellular Biology, Harvard University, Cambridge, MA 02138
Department of Molecular & Cellular Biology, Harvard University, Cambridge, MA 02138
Department of Chemistry & Chemical Biology, Harvard University, Cambridge, MA 02138
Candice J. Sheehan
Department of Molecular & Cellular Biology, Harvard University, Cambridge, MA 02138
In-Sik Kim
BioCARS, Argonne National Laboratory, The University of Chicago, Lemont, IL 60439
BioCARS, Argonne National Laboratory, The University of Chicago, Lemont, IL 60439
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025
Department of Molecular & Cellular Biology, Harvard University, Cambridge, MA 02138
School of Engineering & Applied Sciences, Harvard University, Allston, MA 02134

Notes

1
To whom correspondence may be addressed. Email: [email protected].

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