Mechanism of catalytic regulation by an allosteric hot spot in dihydrofolate reductase
6 affiliations
- 1Laboratoire Chimie Physique et Chimie du Vivant, Department of Chemistry, Ecole Normale Supérieure, Paris Sciences et Lettres, Sorbonne Université, Centre National de la Recherche Scientifique, Paris 75005, France
- 2Gulliver, École supérieure de physique et de chimie industrielles de la ville de Paris - Paris Science et Lettres, CNRS, Paris 75005, France
- 3Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas 75235, TX
- 4T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore 21218, MD
- 5Departamento de Química Física, Universidad de Valencia, Burjassot 46100, Spain
- 6Sorbonne, Université, CNRS, Institut de Biologie Paris-Seine, Laboratoire Jean Perrin, Paris 75005, France
Abstract
Allostery is central to catalysis and regulation, yet how distal perturbations control enzyme activity remains unresolved. Here we elucidate how a distal allosteric hot spot regulates catalysis in dihydrofolate reductase, a benchmark enzyme exhibiting latent allostery. We combine enhanced conformational sampling, reactive free-energy calculations of the chemical step, and perturbation-induced nonequilibrium molecular dynamics with evolutionary analysis and experimental kinetics to obtain a comprehensive molecular description of conformational landscapes, catalytic activity, and allosteric communication. We show that a mutational perturbation at the hot spot redistributes the populations of two globally similar conformations with a subtle but critical structural difference that makes one catalytically competent and the other not. Nonequilibrium relaxation simulations further reveal that the transition between these two states in the mutant proceeds through a stepwise sequence of local structural rearrangements rather than a concerted global motion. Guided by this mechanism, we rationally engineer a compensatory second-site mutation that partially restores activity. These results provide a unified thermodynamic and kinetic view of allostery and establish a general, mechanistically guided framework for engineering allosteric regulation in enzymes.
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BibTeX
@article{guenon2026mechanism,
title = {{Mechanism of catalytic regulation by an allosteric hot spot in dihydrofolate reductase}},
author = {Paul Guénon and Marion Chauveau and Karolina Filipowska and Iñaki Tuñón and Clément Nizak and Kimberly A. Reynolds and Guillaume Stirnemann and Olivier Rivoire and Damien Laage},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2026},
month = oct,
doi = {10.1073/pnas.2610950123},
url = {https://doi.org/10.1073/pnas.2610950123},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Mechanism of catalytic regulation by an allosteric hot spot in dihydrofolate reductase
AU - Paul Guénon
AU - Marion Chauveau
AU - Karolina Filipowska
AU - Iñaki Tuñón
AU - Clément Nizak
AU - Kimberly A. Reynolds
AU - Guillaume Stirnemann
AU - Olivier Rivoire
AU - Damien Laage
PY - 2026
DA - 2026/10/30
JO - Proceedings of the National Academy of Sciences of the United States of America
DO - 10.1073/pnas.2610950123
UR - https://doi.org/10.1073/pnas.2610950123
AB - Allostery is central to catalysis and regulation, yet how distal perturbations control enzyme activity remains unresolved. Here we elucidate how a distal allosteric hot spot regulates catalysis in dihydrofolate reductase, a benchmark enzyme exhibiting latent allostery. We combine enhanced conformational sampling, reactive free-energy calculations of the chemical step, and perturbation-induced nonequilibrium molecular dynamics with evolutionary analysis and experimental kinetics to obtain a comprehensive molecular description of conformational landscapes, catalytic activity, and allosteric communication. We show that a mutational perturbation at the hot spot redistributes the populations of two globally similar conformations with a subtle but critical structural difference that makes one catalytically competent and the other not. Nonequilibrium relaxation simulations further reveal that the transition between these two states in the mutant proceeds through a stepwise sequence of local structural rearrangements rather than a concerted global motion. Guided by this mechanism, we rationally engineer a compensatory second-site mutation that partially restores activity. These results provide a unified thermodynamic and kinetic view of allostery and establish a general, mechanistically guided framework for engineering allosteric regulation in enzymes.
SN - 0027-8424
SN - 1091-6490
ER -