To gain insight into how the mutations influence activity, Rod
and coworkers employed several long (10 ns) molecular dynamics
simulations of M42W-DHFR and wild type. They simulated the
Michaelis complexes using explicit solvent and the 1RX2 crystal
structure. They examined whether the mutations impact correlated
motions and/or the distribution of conformers sampled by the
enzyme and the resulting effects on hydrogen bonds. The authors
quantitated the coupling between residues by calculating the
covariance between the fluctuations of the two residues. The results
indicate that the dynamics of the closed conformation in the M42W
mutant are altered because the mutation disrupts a network of
coordinated motion that promotes hydride transfer [84, 88].
In a study by Mauldin et al., NMR relaxation data was used to
examine the dynamics of M42W-DHFR in the ternary complex
with THF. The authors used a strategy where they measured the
conformational fluctuations of backbone amide and side-change
methyl groups on multiple timescales. They attributed changes in
picosecond to nanosecond dynamics to mutational effects propagating throughout a network of interacting residues and micro- to
millisecond timescale changes to the mutation resulting in an
increased rate of switching in the catalytic core. They observed
two distinct groups of residues that experience R 2 dispersion in
M42W-DHFR: 15 residues within the catalytic core of the protein
and a cluster of 5 residues lining the pABG binding cleft. They
found that conformational switching within the pABG binding
cleft may act to eject THF from M42W-DHFR [83]. In addition,
they proposed that M42 acts as a “dynamic hub” in DHFR by
coordinating motion on multiple timescales and that disrupting
these dynamic interactions may be an effective method of allosterically modulating DHFR function [83].
5.2 Mutations
in the Met20 Loop
The direct role of the Met20 loop in DHFR catalysis was outlined
in Subheadings 2.2 and 2.3. Although the Met20 loop is directly
implicated in the catalytic cycle, some residues are considered distal
because the side chains are oriented toward the solution and do not
contact cofactor or substrate [80]. An investigation of the contribution of the Met20 loop to DHFR catalysis was performed by
constructing a DHFR deletion mutant of four residues in the
Met20 loop (residues 16-19) [89]. Three of these four residues
are considered distal. However, N18 is not considered distal
because it is within contact distance to the cofactor. The deletion
mutant (DL1) resulted in a 400-fold decrease in the rate of hydride
transfer (950 s
À1 for WT enzyme and 1.7 s
À1 for DL1). The K M
and K d values for DHF and NADPH increased, but not drastically.
These observations support Met20 loop acting as an active site gate
that affects the organization of bound substrate and cofactor to
form an active complex [80].
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
205
and coworkers employed several long (10 ns) molecular dynamics
simulations of M42W-DHFR and wild type. They simulated the
Michaelis complexes using explicit solvent and the 1RX2 crystal
structure. They examined whether the mutations impact correlated
motions and/or the distribution of conformers sampled by the
enzyme and the resulting effects on hydrogen bonds. The authors
quantitated the coupling between residues by calculating the
covariance between the fluctuations of the two residues. The results
indicate that the dynamics of the closed conformation in the M42W
mutant are altered because the mutation disrupts a network of
coordinated motion that promotes hydride transfer [84, 88].
In a study by Mauldin et al., NMR relaxation data was used to
examine the dynamics of M42W-DHFR in the ternary complex
with THF. The authors used a strategy where they measured the
conformational fluctuations of backbone amide and side-change
methyl groups on multiple timescales. They attributed changes in
picosecond to nanosecond dynamics to mutational effects propagating throughout a network of interacting residues and micro- to
millisecond timescale changes to the mutation resulting in an
increased rate of switching in the catalytic core. They observed
two distinct groups of residues that experience R 2 dispersion in
M42W-DHFR: 15 residues within the catalytic core of the protein
and a cluster of 5 residues lining the pABG binding cleft. They
found that conformational switching within the pABG binding
cleft may act to eject THF from M42W-DHFR [83]. In addition,
they proposed that M42 acts as a “dynamic hub” in DHFR by
coordinating motion on multiple timescales and that disrupting
these dynamic interactions may be an effective method of allosterically modulating DHFR function [83].
5.2 Mutations
in the Met20 Loop
The direct role of the Met20 loop in DHFR catalysis was outlined
in Subheadings 2.2 and 2.3. Although the Met20 loop is directly
implicated in the catalytic cycle, some residues are considered distal
because the side chains are oriented toward the solution and do not
contact cofactor or substrate [80]. An investigation of the contribution of the Met20 loop to DHFR catalysis was performed by
constructing a DHFR deletion mutant of four residues in the
Met20 loop (residues 16-19) [89]. Three of these four residues
are considered distal. However, N18 is not considered distal
because it is within contact distance to the cofactor. The deletion
mutant (DL1) resulted in a 400-fold decrease in the rate of hydride
transfer (950 s
À1 for WT enzyme and 1.7 s
À1 for DL1). The K M
and K d values for DHF and NADPH increased, but not drastically.
These observations support Met20 loop acting as an active site gate
that affects the organization of bound substrate and cofactor to
form an active complex [80].
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
205