Chapter 12
Distal Regions Regulate Dihydrofolate Reductase-Ligand
Interactions
Melanie Goldstein and Nina M. Goodey
Abstract
Protein motions play a fundamental role in enzyme catalysis and ligand binding. The relationship between
protein motion and function has been extensively investigated in the model enzyme dihydrofolate reductase
(DHFR). DHFR is an essential enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate.
Numerous experimental and computational methods have been used to probe the motions of DHFR
through the catalytic cycle and to investigate the effect of distal mutations on DHFR motions and ligand
binding. These experimental investigations have pushed forward the study of protein motions and their role
in protein-ligand interactions. The introduction of mutations distal to the active site has been shown to have
profound effects on ligand binding, hydride transfer rates and catalytic efficacy and these changes are
captured by enzyme kinetics measurements. Distal mutations have been shown to exert their effects
through a network of correlated amino acids and these effects have been investigated by NMR, protein
dynamics, and analysis of coupled amino acids. The experimental methods and the findings that are
reviewed here have broad implications for our understanding of enzyme mechanisms, ligand binding and
for the future design and discovery of enzyme inhibitors.
Key words Allostery, Protein motions, Dihydrofolate reductase, Point mutation
1 Introduction
The interaction between a ligand and its biomolecular target forms
the basis for many physiological processes [1, 2]. Understanding
the molecular mechanisms by which ligands recognize and bind
their targets remains a fundamental question in biochemistry and
biophysics. Over the years, several models have been proposed.
Early on, the “lock-and-key” model, where a protein and ligand
are a perfect match of rigid complementary structures, dominated
(Fig. 1) [3, 4]. In this model, the protein’s ligand binding site is
assumed to have a single, rigid shape and the ligand distinguishes
between different proteins in the cell based on the different shapes
of their ligand binding sites [4]. As technology has advanced, so has
our understanding of protein structure and dynamics, and it is now
Luisa Di Paola and Alessandro Giuliani (eds.), Allostery: Methods and Protocols, Methods in Molecular Biology, vol. 2253,
https://doi.org/10.1007/978-1-0716-1154-8_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
185
Distal Regions Regulate Dihydrofolate Reductase-Ligand
Interactions
Melanie Goldstein and Nina M. Goodey
Abstract
Protein motions play a fundamental role in enzyme catalysis and ligand binding. The relationship between
protein motion and function has been extensively investigated in the model enzyme dihydrofolate reductase
(DHFR). DHFR is an essential enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate.
Numerous experimental and computational methods have been used to probe the motions of DHFR
through the catalytic cycle and to investigate the effect of distal mutations on DHFR motions and ligand
binding. These experimental investigations have pushed forward the study of protein motions and their role
in protein-ligand interactions. The introduction of mutations distal to the active site has been shown to have
profound effects on ligand binding, hydride transfer rates and catalytic efficacy and these changes are
captured by enzyme kinetics measurements. Distal mutations have been shown to exert their effects
through a network of correlated amino acids and these effects have been investigated by NMR, protein
dynamics, and analysis of coupled amino acids. The experimental methods and the findings that are
reviewed here have broad implications for our understanding of enzyme mechanisms, ligand binding and
for the future design and discovery of enzyme inhibitors.
Key words Allostery, Protein motions, Dihydrofolate reductase, Point mutation
1 Introduction
The interaction between a ligand and its biomolecular target forms
the basis for many physiological processes [1, 2]. Understanding
the molecular mechanisms by which ligands recognize and bind
their targets remains a fundamental question in biochemistry and
biophysics. Over the years, several models have been proposed.
Early on, the “lock-and-key” model, where a protein and ligand
are a perfect match of rigid complementary structures, dominated
(Fig. 1) [3, 4]. In this model, the protein’s ligand binding site is
assumed to have a single, rigid shape and the ligand distinguishes
between different proteins in the cell based on the different shapes
of their ligand binding sites [4]. As technology has advanced, so has
our understanding of protein structure and dynamics, and it is now
Luisa Di Paola and Alessandro Giuliani (eds.), Allostery: Methods and Protocols, Methods in Molecular Biology, vol. 2253,
https://doi.org/10.1007/978-1-0716-1154-8_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
185