Nuclear Magnetic Resonance Study of the Role of M42 in the Solution Dynamics of Escherichia coli Dihydrofolate Reductase

Abstract

It is widely recognized that key positions throughout a protein’s structure contribute unequally to function. In light of recent studies that suggest protein dynamics are required for function, a number of these residues may serve to promote motions required for ligand binding and catalysis. In the present NMR study, the conformational dynamics of the dihydrofolate reductase (DHFR) mutant M42W, in the presence of methotrexate and NADPH, are characterized and compared to the wild-type enzyme. M42 is distal to the active site, yet the M42W substitution regulates catalysis and ligand affinity, and is therefore analogous to an allosteric modulator of DHFR function. To gain understanding of how this mutation regulates activity, we employ a “pandynamic” strategy by measuring conformational fluctuations of backbone amide and side-chain methyl groups on multiple timescales. Changes in ps-ns dynamics indicate that the mutational effects are propagated throughout a network of interacting residues within DHFR, consistent with a role for M42 as a dynamic communication hub. On the μs-ms timescale, mutation increases the rate of switching in the catalytic core. Mutation also introduces switching in the adenosine binding subdomain that occurs at a higher frequency than in the catalytic core, and which correlates with the rate of product release for M42W-DHFR. Finally, a structurally inferred analysis of side-chain dynamics suggests that the M42W mutation dampens motional contributions from non-local effects. These data show that the M42W mutation alters the dynamics of DHFR and are consistent with theoretical analysis that suggests the mutation disrupts motion that promotes catalysis.

In Vitro Phosphorylation of the Focal Adhesion Targeting Domain of Focal Adhesion Kinase by Src Kinase

“…RDCs provide long-range orientation information on the NH bond vector and are often used to assess structural perturbations upon mutation (50) and ligand binding (51). We chose pH 6.0 because the solution structure of the FAT domain had been solved at this pH (19).…”

Dynamically committed, uncommitted, and quenched states encoded in protein kinase A revealed by NMR spectroscopy

“…In fact, recent studies on dihydrofolate reductase (56), triosephosphate isomerase (57), ribonuclease A (58), HIV-1 reverse transcriptase (59), and imidazole glycerol phosphate synthase (60) show that slow microsecond to millisecond motions are severely dampened when these enzymes are inhibited (56,59,60) or when catalytically hindering mutations are introduced (57,58).…”

Connecting Protein Conformational Dynamics with Catalytic Function As Illustrated in Dihydrofolate Reductase

“…(20) We do not observe this elongated ADL in the double mutation. If anything, there seems to be a slight compression both in the Michaelis complex and in the transition state since both mutations introduce steric congestions propagated to the active site,(21-22) especially in the M42W mutation site(28) (see below).…”

“…(18-19, 21-22, 68-70) Mauldin et al showed that the M20 loop adopts the closed conformation in the ternary complex with NADPH and methotrexate both in the M42W and G121V mutants. (21-22) While the G121V mutantion results in a large response on the M42 dynamic fluctuations from NMR relaxation experiments,(21) the M42W mutation showed that the M42 site serves has a hob connecting the dynamic fluctuations throughout the protein. (22) The M20 conformational fluctuations in the wt-DHFR and single and double mutants at M42 and G121 as well as their coupled motions in the Michaelis complex have been examined by Brooks and coworkers.…”