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

Abstract

The characterization of conformational changes that drive induced-fit mechanisms and their quantitative importance to enzyme specificity are essential for a full understanding of enzyme function. Here, we report on M.HhaI, a sequence-specific DNA cytosine C 5 methyltransferase that reorganizes a flexible loop (residues 80 -100) upon binding cognate DNA as part of an induced-fit mechanism. To directly observe this ϳ26 Å conformational rearrangement and provide a basis for understanding its importance to specificity, we replaced loop residues Lys-91 and Glu-94 with tryptophans. The double mutants W41F/K91W and W41F/E94W are relatively unperturbed in kinetic and thermodynamic properties. Ligand-induced changes in protein conformation can contribute to enzyme catalysis, regulation of function, and substrate specificity. Induced-fit mechanisms involve conformational rearrangements of an enzyme to facilitate or enhance correct substrate binding and can provide improved specificity (1-3). First introduced in 1958 (4), induced-fit mechanisms contribute to diverse biological processes (5), including tRNA binding in ribosomes (6), DNA-modifying enzymes (7-9), kinases (10, 11), RNA folding (12), DNA binding specificity (13,14), polymerases (15, 16), and many others. However, direct evidence for an induced-fit mechanism through the observation of real-time protein conformational rearrangements coupled to specificity have been quantitated for a small number of enzymes (17)(18)(19)(20).Enzymes that sequence-specifically modify DNA, including nucleases, repair enzymes, and methyltransferases are faced with severe challenges of substrate recognition and specificity due to the overwhelming abundance of sites that are closely related to the cognate sequence (1,3,5,21). Mechanisms posited to account for this discrimination are diverse and often require an induced-fit process as the enzyme-DNA complex moves from a nonspecific site to the cognate sequence (22, 23). The availability of high resolution structures of cognate DNAenzyme complexes for many such systems provides a detailed understanding of specific interactions leading to tight and cognate binding (24 -27). However, interactions leading to nonspecific substrate binding, which facilitate site searching, are far less characterized (1, 3, 28). Furthermore, the interconversion of conformers as the enzyme goes from nonspecific to cognate sites prior to forming the catalytically competent complex can contribute to such specificity (13, 21, 28 -31).The DNA cytosine C 5 methyltransferase M.HhaI binds DNA substrates between its two domains and the cofactor AdoMet 2 in the large domain near the active site (see Fig. 1). Inspection of two cocrystal structures of M.HhaI, one involving the cognate DNA and the cofactor product AdoHcy (3MHT.pdb) (24), the other involving nonspecific DNA and AdoHcy (2HMY.pdb) (32), suggest that the enzyme may exploit an induced-fit mechanism. Induced-fit DNA binding was first proposed for M.HhaI in 1994 when the first ternary complex with cognate DNA, cof...

Coupling Sequence-specific Recognition to DNA Modification

“…Mutant Construction-Four mutants of WT M.HhaI were constructed, R240A, W41F, W41F/K91W, and W41F/E94W as previously reported (12). Briefly, PCR mutagenesis was done using the QuikChange kit (Stratagene).…”

“…Stopped-flow Fluorescence-Time-resolved measurements were made at 22°C with excitation at 290 nm and a cut-off filter at 295 nm similar to protocols previously reported (12). Briefly, 4-mm slit widths were used on an Applied Photophysics SX.18MV stopped-flow reaction analyzer equipped with a single channel emission photomultiplier tube positioned 90°from the excitation beam.…”

“…Although the cofactor alone has no impact on loop positioning (12), rate constants of loop closure with AdoMet were faster than those with AdoHcy when in the presence of COG DNA, (Fig. 2D and supplemental Table S2).…”

“…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).…”

“…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). Loop positioning and the interconversion between the open and closed conformers, as determined from the intensities and rates of change in fluorescence signal are highly dependent on DNA sequence and confirm that cognate DNA stabilizes the loopclosed conformer whereas nonspecific DNA stabilizes the open conformer.…”