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. 2003 Jun 10;100(12):6980-5.
doi: 10.1073/pnas.1230801100. Epub 2003 May 19.

Correlated motion and the effect of distal mutations in dihydrofolate reductase

Thomas H Rod  1 , Jennifer L Radkiewicz, Charles L Brooks 3rd
Affiliations

Correlated motion and the effect of distal mutations in dihydrofolate reductase

Thomas H Rod et al. Proc Natl Acad Sci U S A. .

Abstract

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme [Rajagopalan, P. T. R, Lutz, S., and Benkovic, S. J. (2002) Biochemistry 41, 12618-12628]. On the basis of extensive computer simulations for wild-type DHFR from Escherichia coli and four mutants (G121S, G121V, M42F, and M42F/G121S), we show that key parameters for catalysis are changed. The parameters we study are relative populations of different conformations sampled and hydrogen bonds. We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme. Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations. We finally examine the role a structural perturbation will have on the hydride transfer step. On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks.

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Figures

Fig. 1.
Fig. 1.
The catalytic cycle for the reduction of H2F to H4F by DHFR as published in refs. and .
Fig. 2.
Fig. 2.
The structure for DHFR from E. coli in the closed conformation (PDB ID: IRX2) with NADP+ and folate bound to the enzyme. The locations of residues 42 and 121 from Table 1 are illustrated by solid balls. The structure was generated by using molscript (27). The panel below the structure illustrates the three primary loop conformations observed in crystal structures from PDB entries 1RX2, 1RD7 (B-chain), and 1RX7. (Right) The relevant chemical species and sites of reactivity are shown. The atoms where the proton and hydride are added to form H4F are indicated by arrows.
Fig. 3.
Fig. 3.
Covariance matrix for the fluctuations of the Cα atoms in wild-type DHFR (WT) and various mutants. Yellow and red regions indicate that the Cα atoms move in a concerted way (positively correlated movements), and dark blue means they move opposite to each other (anticorrelated movements). The scale goes from –0.6 (dark blue) to 1 (red). We note that we get the same qualitative picture for the correlated motions if all heavy atoms are included in the calculation of the covariance matrix.
Fig. 4.
Fig. 4.
(Left) Shown are the energy levels for different conformations of the M20 loop sampled in the simulations of native and mutant Michaelis complexes of DHFR, relative to the closed conformation. Each color represents a particular mutant, and the five different conformations found from our cluster analysis are shown along the horizontal axis. The energies are the average energies calculated by using a generalized Born implicit solvent model from snapshots from the portions of the trajectories belonging to particular loop conformations. The error bars represent the standard errors about these averages. The arrows indicate the progression in time. (Right) Shown is a representative trajectory in ψ–ι space for loop residue Gly-17 in the G121V mutant to illustrate the extent to which different conformations may be differentiated.
Fig. 5.
Fig. 5.
(Right) Shift in activation free energy, formula image, for forward hydride transfer is plotted against the reaction free energy ΔGi→f. The free energy differences plotted are defined in Left. All calculated energies are based on the rates, reported in ref. , for forward and reverse hydride transfer, and the pKa values for the protonation step. Data depicted by red points are based on the rates combining protonation and hydride transfer, i.e., they are pH dependent, whereas data depicted by blue points are based on rates solely for the hydride transfer step. These values are calculated from the pKa values and pH-dependent rates determined by Rajagopalan et al. (8) and given in Table 1. No pKa values have been determined for the two mutants where Met-42 is replaced with Phe. The green dotted lines follow the points for mutation of Met-42 to either Phe or Trp in the wild-type enzyme and in the G121S mutant.
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