The Coupling of Tight DNA Binding and Base Flipping

Abstract

The molecular basis of enzymatic catalysis is of broad interest, with implications for biocatalyst design and drug development. The abundance of detailed three-dimensional structures and investigational methods provides newly addressable aspects of enzymatic function. We are interested in the importance of protein motion, and particularly correlated motions, to catalysis. The underlying premise is that protein-solvent interactions are converted into peptide motions, resulting in the transient stabilization of active site elements with preferred reactivities (1, 2).Recent studies (1) have provided highly suggestive evidence for this concept. Molecular dynamics investigations of dihydrofolate reductase demonstrate that strong coupled motions in the reactive complex disappear in the product complexes, indicating that these motions may be linked to catalysis. Mutants that alter the kinetics of particular catalytic steps are concentrated within segments of the protein structure shown to participate in highly correlated motions (1). Solid state NMR and solution NMR relaxation studies have measured substrate and protein dynamics that are matched to the turnover time of the respective enzymes (3). Studies of hydrogen and electron tunneling during enzyme catalysis provide further evidence for the importance of protein dynamics to catalytic events at the active site (4, 5).Molecular dynamic simulations of catechol O-methyltransferase and M.HhaI 1 DNA methyltransferase provided initial evidence for correlated motions within the active sites of these enzymes (6, 7). We sought to test the importance to catalysis of motions made by specific distal residues (His 127 -Thr 132 ) in facilitating active site chemistries by altering the position and orientation of critical residues such as Val 121 . Alanine scan point mutagenesis and kinetic characterization of individual steps in the catalytic cycle were used to probe the effects of such mutations and provide insights into the roles of correlated motions. M.HhaI provides an excellent structurally and functionally tractable enzyme to study various aspects of catalysis, including base flipping and the importance of motions to catalysis. M.HhaI, from Haemophilus haemolyticus, is an AdoMetdependent C 5 -cytosine methyltransferase that methylates the central cytosine (C) in the recognition sequence 5Ј-GCGC-3Ј after stabilizing the target base in an extrahelical position. Many M.HhaI crystal structures provide structural insights into the mechanisms of DNA methylation and base flipping (8). Functional analysis of the WT M.HhaI has been extensive (9 -12), including K D DNA determination for a variety of DNA substrates (13,14). Many structural components of the M.HhaI mechanism have been examined by mutagenesis including Gln 237 , which positions itself into the DNA helix and interacts with the lone guanine (15), and Cys 81 , which forms a covalent bond to the target cytosine (16). Other mutational studies have examined protein-phosphate interactions (17) and conserved residues within the...

Statistical coevolution analysis and molecular dynamics: Identification of amino acid pairs essential for catalysis

“…Furthermore, adjacent alanine mutants that were not highlighted by our SCA⅐MD approach (Asp-128 3 Ala, Asn-129 3 Ala, Gly-130 3 Ala, Asn-131 3 Ala, and Thr-132 3 Ala) show no change in methyl transfer (Fig. 4D) (39). Remarkably, mutation of Asp-73, which is Ϸ21 Å away from the AdoMet cofactor, to alanine (Asp-73 3 Ala) results in a 24-fold increase in AdoMet affinity (40).…”

“…This spatial arrangement of evolutionarily coupled anticorrelated pairs is consistent with previous proposals that methyltransferases use some form of active-site compression to facilitate catalysis (25)(26)(27). The focal point encompasses the target cytosine such that the -angle defined by the glycosidic bond could readily be altered, and prior studies have shown this angle to be highly correlated to catalysis (25,26,39). Also, the focal point is located on the periphery of the large domain, Ϸ10 Å away from its center of mass.…”

“…4B), and, most notably, the majority do not occur within or between well formed secondary structures. Mutation of one such residue to alanine, His-127 3 Ala, which is positioned on the protein surface Ϸ15 Å from the active site, results in a 10-fold decrease in methyl transfer rates (39). Furthermore, adjacent alanine mutants that were not highlighted by our SCA⅐MD approach (Asp-128 3 Ala, Asn-129 3 Ala, Gly-130 3 Ala, Asn-131 3 Ala, and Thr-132 3 Ala) show no change in methyl transfer (Fig.…”

Observing an Induced-fit Mechanism during Sequence-specific DNA Methylation

“…All substrate concentrations were spectrophotometrically determined using Beer's Law and calculated extinction coefficients (37). Determination of k cat -Steady-state product formation over time was monitored at 4°C using a filter binding assay previously described (38) to determine observed k cat values (see Table 1). These values are only observed k cat values, because substrate concentrations were not fully saturating; all enzymes were measured under identical conditions.…”

Coupling Sequence-specific Recognition to DNA Modification

“…1A). Populating the closed conformer of the loop is essential for tight DNA binding and stabilizing the target cytosine that is flipped out of the DNA duplex (11)(12)(13). Using stopped-flow fluorescence spectroscopy to monitor the environment of tryptophan (Trp) residues inserted into the catalytic loop, we recently observed reorganization of this loop upon DNA binding in the absence of cofactor using the M.HhaI mutants W41F, W41F/K91W, and W41F/E94W (12).…”