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How adding a single methylene to dihydrofolate reductase can change its conformational dynamics.

ABSTRACT: Studies of the effects of pressure on proteins from piezophilic (pressure-loving) microbes compared with homologous proteins from mesophilic microbes have been relatively rare. Interestingly, such studies of dihydrofolate reductase show that a single-site mutation from an aspartic acid to a glutamic acid can reverse the pressure-dependent monotonic decrease in activity to that in a monotonic pressure-dependent activation. This residue is near the active site but is not thought to directly participate in the catalytic mechanism. Here, the ways that addition of one carbon to the entire protein could lead to such a profound difference in pressure effects are explored using molecular dynamics simulations. The results indicate that the glutamate changes the coupling between a helix and the β-sheet due to the extra flexibility of the side chain, which further changes correlated motions of other regions of the protein.

SUBMITTER: Penhallurick RW

PROVIDER: S-EPMC8068499 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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How adding a single methylene to dihydrofolate reductase can change its conformational dynamics.

Penhallurick Ryan W RW Harold Alliyah A Durnal Maya D MD Ichiye Toshiko T

The Journal of chemical physics 20210401 16

Studies of the effects of pressure on proteins from piezophilic (pressure-loving) microbes compared with homologous proteins from mesophilic microbes have been relatively rare. Interestingly, such studies of dihydrofolate reductase show that a single-site mutation from an aspartic acid to a glutamic acid can reverse the pressure-dependent monotonic decrease in activity to that in a monotonic pressure-dependent activation. This residue is near the active site but is not thought to directly partic ...[more]

PMID: 33940851

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Project description:The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of (3)JCγCO and (3)JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as it proceeds through its enzymatic cycle, but that ligand flux is brokered by more subtle conformational changes that depend on the side chain motions of critical residues.

Dataset Information

How adding a single methylene to dihydrofolate reductase can change its conformational dynamics.

Publications

How adding a single methylene to dihydrofolate reductase can change its conformational dynamics.

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