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Side-chain conformational heterogeneity of intermediates in the Escherichia coli dihydrofolate reductase catalytic cycle.

ABSTRACT: Escherichia coli dihydrofolate reductase (DHFR) provides a paradigm for the integrated study of the role of protein dynamics in enzyme function. Previous studies of backbone and side chain dynamics have yielded unprecedented insights into the mechanism by which DHFR progresses through the structural changes that occur during its catalytic cycle. Here we report a comprehensive study of the ?1 rotamer populations of the aromatic and ?-methyl containing residues for complexes of the catalytic cycle, based on NMR measurement of (3)JC?CO and (3)JC?N coupling constants. We report conformational and dynamic information for eight distinct complexes, where transitions between rotamer wells may occur on a broad picosecond to millisecond time scale. This large volume of (3)J data has allowed us to fit new Karplus parameterizations for aromatic side chains and to select the best available of previously determined parameters for Ile, Thr, and Val. The (3)JC?CO and (3)JC?N coupling constants are found to be extremely sensitive measures of side chain ?1 rotamers and to give important insights into the extent of conformational averaging. For a subset of residues in DHFR, the extent of rotamer averaging is invariant to the nature of the bound ligand, while for other residues the rotamer averaging differs in one or more complexes of the enzymatic cycle. These variable-averaging residues are generally located near the active site, but the phenomenon extends into the adenosine binding domain. For several residues, the rotamer populations in different DHFR complexes appear to depend on whether the complex is in the closed or occluded state, and some residues are exquisitely sensitive to small changes in the nature of the bound ligand.

SUBMITTER: Tuttle LM

PROVIDER: S-EPMC3796204 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Side-chain conformational heterogeneity of intermediates in the Escherichia coli dihydrofolate reductase catalytic cycle.

Tuttle Lisa M LM Dyson H Jane HJ Wright Peter E PE

Biochemistry 20130507 20

Escherichia coli dihydrofolate reductase (DHFR) provides a paradigm for the integrated study of the role of protein dynamics in enzyme function. Previous studies of backbone and side chain dynamics have yielded unprecedented insights into the mechanism by which DHFR progresses through the structural changes that occur during its catalytic cycle. Here we report a comprehensive study of the χ1 rotamer populations of the aromatic and γ-methyl containing residues for complexes of the catalytic cycle ...[more]

PMID: 23614825

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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.

Project description:Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecDHFR.

Project description:Dihydrofolate reductase (DHFR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F). Because of the absence of any ionizable group in the vicinity of N5 of dihydrofolate it has been proposed that N5 could be protonated directly by a water molecule at the active site in the ternary complex of the Escherichia coli enzyme with cofactor and substrate. However, in the X-ray structures representing the Michaelis complex of the E. coli enzyme, a water molecule has never been observed in a position that could allow protonation of N5. In fact, the side chain of Met 20 blocks access to N5. Energy minimization reported here revealed that water could be placed in hydrogen bonding distance of N5 with only minor conformational changes. The r.m.s. deviation between the conformation of the M20 loop observed in the crystal structures of the ternary complexes and the conformation adopted after energy minimization was only 0.79 A. We performed molecular dynamics simulations to determine the accessibility by water of the active site of the Michaelis complex of DHFR. Water could access N5 relatively freely after an equilibration time of approximately 300 psec during which the side chain of Met 20 blocked water access. Protonation of N5 did not increase the accessibility by water. Surprisingly the number of near-attack conformations, in which the distance between the pro-R hydrogen of NADPH and C6 of dihydrofolate was less than 3.5 A and the angle between C4 and the pro-R hydrogen of NADPH and C6 of dihydrofolate was greater than 120 degrees, did not increase after protonation. However, when the hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the side chain of Met 20 moved away from N5 after approximately 100 psec thereby providing water access. The average time during which water was found in hydrogen bonding distance to N5 was significantly increased. These results suggest that hydride transfer might occur early to midway through the reaction followed by protonation. Such a mechanism is supported by the very close contact between C4 of NADP+ and C6 of folate observed in the crystal structures of the ternary enzyme complexes, when the M20 loop is in its closed conformation.

Dataset Information

Side-chain conformational heterogeneity of intermediates in the Escherichia coli dihydrofolate reductase catalytic cycle.

Publications

Side-chain conformational heterogeneity of intermediates in the Escherichia coli dihydrofolate reductase catalytic cycle.

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