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Originally published In Press as doi:10.1074/jbc.M402950200 on May 12, 2004

J. Biol. Chem., Vol. 279, Issue 30, 31419-31428, July 23, 2004
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The Coupling of Tight DNA Binding and Base Flipping

IDENTIFICATION OF A CONSERVED STRUCTURAL MOTIF IN BASE FLIPPING ENZYMES*

R. August Estabrook, Rebecca Lipson, Ben Hopkins, and Norbert Reich{ddagger}

From the Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106

Received for publication, March 16, 2004 , and in revised form, May 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Val121 is positioned immediately above the extrahelical cytosine in HhaI DNA C5-cytosine methyltransferase, and replacement with alanine dramatically interferes with base flipping and catalysis. DNA binding and kcat are decreased 105-fold for the Val121 -> Ala mutant that has a normal circular dichroism spectrum and AdoMet affinity. The magnitude of this loss of function is comparable with removal of the essential catalytic Cys81. Surprisingly, DNA binding is completely recovered (increase of 105-fold) with a DNA substrate lacking the target cytosine base (abasic). Thus, interfering with the base flipping transition results in a dramatic loss of binding energy. Our data support an induced fit mechanism in which tight DNA binding is coupled to both base flipping and protein loop rearrangement. The importance of the proximal protein segment (His127–Thr132) in maintaining this critical interaction between Val121 and the flipped cytosine was probed with single site alanine substitutions. None of these mutants are significantly altered in secondary structure, AdoMet or DNA affinity, kmethylation, kinactivation, or kcat. Although Val121 plays a critical role in both extrahelical base stabilization and catalysis, its position and mobility are not influenced by individual residues in the adjacent peptide region. Structural comparisons with other DNA methyltransferases and DNA repair enzymes that stabilize extrahelical nucleotides reveal a motif that includes a positively charged or polar side chain and a hydrophobic residue positioned adjacent to the target DNA base and either the 5'- or 3'-phosphate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.HhaI1 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 (His127–Thr132) in facilitating active site chemistries by altering the position and orientation of critical residues such as Val121. 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 AdoMet-dependent C5-cytosine methyltransferase that methylates the central cytosine (C) in the recognition sequence 5'-GCGC-3' 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 KDDNA determination for a variety of DNA substrates (13, 14). Many structural components of the M.HhaI mechanism have been examined by mutagenesis including Gln237, which positions itself into the DNA helix and interacts with the lone guanine (15), and Cys81, which forms a covalent bond to the target cytosine (16). Other mutational studies have examined protein-phosphate interactions (17) and conserved residues within the AdoMet binding pocket (18). Crystal structures have also been solved for the enzyme bound to its cofactor and enzyme bound to DNA containing modified target bases, and mutant structures have been solved (17, 19–21). These studies have revealed a large loop movement involving residues 80–99 which appears to be induced by binding the enzyme to the cognate DNA sequence. The binary complex with a nonspecific DNA sequence has this loop positioned in the open conformation (22), seen also in the binary enzyme-AdoMet complex (20). Moreover, this loop is seen in the closed conformation in the co-crystal structure containing the tightly bound cognate DNA (19); thus, the enzyme appears to follow an induced fit mechanism in which tight DNA binding is coupled to loop movement to the closed conformation and, coincidentally, base flipping (14, 19).

Extrahelical nucleoside stabilization, or base flipping, is used by enzymes to assist in chemical modification of DNA substrates. Enzyme-DNA interactions mediate processes that allow for destabilization of the DNA helix and extrahelical stabilization of the target base. This shift in DNA conformation is utilized in a variety of biological processes, including DNA methylation and DNA repair (23–26), by providing access to buried functionalities on DNA bases that would be inaccessible in a B-form DNA helix. Current proposals on the mechanisms by which enzymes facilitate base flipping include enzymatic manipulations to either 5'- or 3'-phosphates (8, 27), "pinch-pull-push" mechanisms (28), and passive mechanisms involving stabilization of transiently flipped nucleotides in B-form DNA (29). Studies on M.HhaI aimed at elucidating the base flipping mechanism include crystallographic studies (19), theoretical calculations (30), NMR studies (31), mutagenesis (32, 33), and kinetic characterization (14, 34). Many fundamental aspects of base flipping remain highly debated, including which DNA groove the target base moves through as it flips and which protein-DNA contacts facilitate this process.

We initiated this study to investigate the importance of interactions between Val121 and the extrahelical cytosine, and to identify distal protein elements that provide both static and dynamic scaffolding for this interaction. Our motivation came in part from molecular dynamics simulations that identified anti-correlated active site motions (2, 6, 7). This anticorrelated motion could cause the active site to undergo a compression, which was previously proposed to be important for AdoMet-dependent methyl transfer reactions (35). Moreover, we hypothesized that such motions could be disrupted by amino acid substitutions in critical peptide elements. Residues 121–132 originate at the active site with Val121 and end on the protein surface with Thr132 (see Fig. 1). In-between these two residues, M.HhaI makes an unusual {alpha}{beta}-turn with multiple hydrogen bonds including an internal hydrogen bond between His127 and Thr132 (see Fig. 1C). We used alanine substitutions of residues distal from the active site (His127–Thr132) but connected to a critical residue in the active site (Val121) to probe for correlated motions. Val121 is located directly over the target cytosine, and we show its positioning is crucial for base flipping and catalysis.



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  		FIG. 1.
A, M.HhaI (gray) is shown complexed to AdoHcy (yellow) and a DNA substrate (blue). The flipped cytosine (dark blue) can be seen, and red regions mark locations of alanine mutants. B is the same figure as A but with a 90° rotation to look down the axis of the DNA. C shows various contacts (dotted black lines) within the active site of the M.HhaI-DNA complex. DNA is blue; Val121, His127, and Thr132 are shown in red, and Asp128–Asn131 backbones are in orange. Distances are shown from the Val121 side chain to the extrahelical cytosine and 5'-phosphate and from Thr132 to His127.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis, Protein Expression, and Purification— Seven M.HhaI mutants were produced (Val121 -> Ala, His127 -> Ala, Asp128 -> Ala, Asn129 -> Ala, Gly130 -> Ala, Asn131 -> Ala, and Thr132 -> Ala) using the QuikChange PCR mutagenesis kit (Stratagene) with the vector pHSHW-5 (provided by S. Kumar, New England Biolabs) as a template and seven sets of PCR primers (Genset Oligos). PCR products were digested (DpnI) to remove any WT plasmid, transformed via heat shock into competent Escherichia coli strain ER1727, and plated onto agar plates supplemented with 60 µg/ml ampicillin. Sequencing of DNA from individual colonies was done by the University of Illinois, Urbana-Champaign Biotechnology Center (Urbana, IL). WT and mutant M.HhaI overexpression from pHSHW-5 derived vectors was accomplished with early log phase (A600 = 0.4) induction with 1.0 mM isopropyl-{beta}-D-thiogalactopyranoside. Cells were centrifugally harvested, and protein was purified according to methods published previously (36). After purification, enzymes were >95% pure and were flash-frozen in 20-µl aliquots and stored at –70 °C. WT protein concentration was determined by active site titration, and mutant protein concentration was by Bradford staining based on a standard curve of the WT protein (37).

Confirmation of Mutant M.HhaI Sequence by Mass Spectrometry— Nano-electrospray ionization was used to confirm the substitution of alanine at selected positions in M.HhaI. Both mutant and WT enzymes were run on SDS-PAGE gels and visualized by silver-staining (Silver-Quest, Invitrogen). Protein bands were excised from the gel and subsequently destained in 1.5-ml siliconized Eppendorf tubes. To enhance diffusion of protease into the gel matrix, gel pieces were dried by speed-vac (Savant) and incubated with 100 µl of 12.5 ng/µl sequencing grade modified trypsin (Promega) for 20–30 min on ice after which excess protease was removed and replaced with 50 mM NH4HCO3 for overnight digestion at 37 °C. The resulting peptides were extracted several times with a 50% acetonitrile, 5% formic acid solution, lyophilized, and resuspended in 5% formic acid. Digested peptides were desalted and concentrated using POROS R2 resin (Perceptive Biosystems) that had been loaded into a nanospray capillary (Proxeon Biosystems). Peptides were loaded onto the resin, washed with 5% formic acid, and eluted into a borosilicate capillary (Proxeon Biosystems) with 50% methanol, 5% formic acid. The capillary containing the eluted peptides was loaded onto an Applied Biosystems/MDS Sciex Q-STAR quadrupole/time-of-flight mass spectrometer, and data were collected over a mass range m/z 300–1500 in positive ion mode. Peptide peaks containing the desired substituted amino acids were observed for all mutants but not for the WT protein, and similarly, the WT peptide was observed in WT protein and not in the mutants. Both WT and mutant peptide were analyzed further by tandem mass spectrometry (MS/MS) for its amino acid sequence. Data (not shown) were collected over a mass range of m/z 50–2000 in positive ion mode.

Circular Dichroism—Circular dichroism was performed on WT and mutant M.HhaI in 50 mM sodium phosphate, pH 7.0, at room temperature. Data were collected on an Aviv 202 Circular Dichroism Spectrophotometer using a 500-µl quartz fluorescence cuvette with a 0.2-cm slit width (Starna). Data were collected between 190 and 265 nm.

Oligonucleotide Synthesis and Purification—Oligonucleotides used for kinetic analysis required the use of multiple DNA substrates with a single recognition site but with variations at the target base. The variations are shown below with the recognition sequence underlined and the target bases in boldface. C is cytosine; M is 5-methylcytosine; F is 5-fluorocytosine, and B is an abasic nucleotide. Abox, 5'-GGGAATTCATGGCGCAGTGGGTGGATCCAG-3', and 3'-CCCTTAAGTACCGCGTCACCCACCTAGGTC-5'; FAMbox, 5'-GGGAATTCATGGFGCAGTGGGTGGATCCAG-3', and 3'-CCCTTAAGTACCGMGTCACCCACCTAGGTC-5'; ABMbox, 5'-GGGAATTCATGGBGCAGTGGGTGGATCCAG-3', and 3'-CCCTTAAGTACCGMGTCACCCACCTAGGTC-5'.

Substrate oligonucleotides were synthesized by Midlands DNA (Midland, TX) and purified on a Dynamax PureDNA high pressure liquid chromatography column (Rainin Instrument Co.) according to the manufacturer's specifications. Oligonucleotides were stored in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). Concentrations were photometrically determined by using calculated extinction coefficients (9). Oligonucleotide duplexes were formed by annealing the single strands in TE buffer with 10 mM NaCl and heated to 95 °C for 5 min before being cooled slowly to room temperature. Ratios of single strands were optimized to provide the most duplex with the least amount of residual single strands. For gel mobility shift assays, DNA substrates were radiolabeled by using [{gamma}-32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (New England Biolabs).

Cofactors—AdoMet and AdoHcy were purchased from Sigma and stored in 0.1 N HCl.

Steady State Assays—kcat values for WT and mutant enzymes were determined as reported previously (9) by using filter binding assays with a few variations. This turnover constant, and all other reported kcat values, is actually an apparent constant because it was determined at a single concentration of one or both of the two substrates. Reactions were initiated by the addition of enzyme (10 and 20 nM) to AdoMet (1.2 µM) and Abox DNA (1 µM) in MR buffer (100 mM Tris, pH 8.0, 10 mM EDTA, 10 mM DTT, 0.2 mg/ml BSA) incubated at 37 °C. Six time points were taken at 10-s intervals, quenched on DE-81 filters, and processed as described previously (37). Samples were analyzed with a Beckman Coulter LS6500 MultiPurpose Scintillation Counter, and linear graphs were fit using Kaleidagraph.

kcat values were derived for WT and Val121 -> Ala M.HhaI by another filter binding, steady state assay. Reactions were initiated by the addition of enzyme (8 µM for Val121 -> Ala and 100 pM for WT) to AdoMet (125 nM for WT and 10 µM for Val121 -> Ala) and Abox DNA (100 nM for WT and 10 µM for Val121 -> Ala) in PD buffer (20 mM potassium phosphate, pH 7.5, 200 mM NaCl, 0.2 mM EDTA, 0.2 mg/ml BSA, 2 mM DTT, 10% glycerol) incubated at 37 °C. Time points were taken every 10 min for 1 h.

AdoMet Equilibrium Dissociation Constant—KDAdoMet values were determined for WT and mutant enzymes by using native protein fluorescence as described previously (9). Briefly, an LS50B luminescence spectrometer (PerkinElmer Life Sciences) was used for fluorescence measurements at room temperature. Excitation slit width was 5 mm, and emission slit width was 7.5 mm. A xenon lamp was used to excite at a wavelength of 280 nm. Emission spectra were recorded from 320 to 430 nm at a scan speed of 100 nm/min. The samples contained enzyme (1 µM), 100 mM Tris, pH 8.0, 10 mM EDTA, 10 mM DTT, and varying AdoMet concentrations (0 to 200 µM). For obtaining KDAdoMet values, areas of the spectral curves were determined using Origin, and curves were fit using SigmaPlot. The equation was fit to a rectangular hyperbola.

Single Turnover Assays—Single turnover assays were conducted as reported previously (9) with two different DNA substrates, Abox DNA and FAMbox DNA. For Abox DNA single turnover assays, AdoMet (3 µM) and Abox DNA (100 nM) were mixed in MR buffer and incubated at 6 °C. Reactions were initiated by the addition of excess enzyme (0.4 to 3 µM), and time points were taken at 4, 8, 12, 16, 25, 40, and 60 s, quenched in a final concentration of 0.5% SDS, and spotted on DE-81 filters for analysis. For FAMbox DNA single turnover assays, AdoMet (800 nM) and enzyme (750 nM) in PD buffer were incubated at 37 °C. Reactions were initiated by FAMbox DNA addition (300 nM). Time points taken at 2, 4, 10, 30, 70, and 120 min were spotted on DE-81 filters for quenching. SigmaPlot was used to fit all single turnover exponential rise to maximum curves.

DNA Equilibrium Dissociation Constant—KDDNA values were determined as reported previously (9) in the presence of the cofactor AdoHcy for two different DNA substrates, Abox and ABMbox. For KDAbox-DNA, AdoHcy (25 µM), radiolabeled Abox DNA (50 pM), and enzyme (10 pM to 1 nM) were used. For the Val121 -> Ala M.HhaI Abox DNA gel shift, enzyme concentrations up to 4 µM were used. For KDABMbox-DNA, AdoHcy (25 µM), radiolabeled ABMbox DNA (200 pM), and enzyme (1 pM to 5 nM) were used. Samples were incubated at room temperature for 10 min in MR buffer and then loaded onto a pre-run, 12% nondenaturing polyacrylamide gel. Gels were run at 300 V for 150 min at room temperature. Gels were exposed to image plates and analyzed using a Storm 840 densitometer (Amersham Biosciences). Densitometry was performed using ImageQuant (Amersham Biosciences). SigmaPlot was used to fit the data to a rectangular hyperbola curve and obtain KDDNA values.

Superimpositions and Computer-generated Graphics—Images of M.HhaI were generated using Pymol, SYBYL, and InsightII on Silicon Graphics computers. Protein Data Bank structures of M.HhaI, M.HaeIII, M.TaqI, and DNA glycosylases include codes 1MHT [PDB] -9MHT, 1HMY [PDB] , 1DCT [PDB] , 1G38 [PDB] , 1BNK [PDB] , 1EBM [PDB] , 1DIZ [PDB] , and 1EMH [PDB] . Superimpositions for Fig. 10C were done on InsightII by using the Transpose function with six sets of defining atoms: C-3' and C-4' carbon atoms of the flipped sugar, two phosphate atoms flanking the flipped base, and side chain {alpha}-carbons for Val121 and Arg165 (Val111 and Arg155 for M.HaeIII). Superimpositions that had no DNA atoms, 1HMY [PDB] , used just the protein side chain {alpha}-carbons. All structures were superimposed onto 3MHT [PDB] .



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  		FIG. 10.
A, the active site of M.HhaI is shown with Arg165, Cys81, Glu119, flipped cytosine, and AdoHcy. The image was generated on InsightII from 3MHT [PDB] .pdb, and hydrogen bonds are shown (dotted line) with distances. The Cys81 thiolate interaction with C6 of the cytosine and the C5 interaction of cytosine with the co-factor are also shown. B was constructed on Isis Draw and shows the AdoMet cofactor, many active site interactions (dotted lines), and the {chi} angle or propeller twist that rotates around the glycosidic bond. Improper {chi} angles can disrupt many of the interactions shown in A and B. C shows superimpositions of Val121, Arg165, and flipped residues for various WT M.HhaI structures and one M.HaeIII structure. M.HaeIII residues, Val111 and Arg155, are shown in parentheses. Red is the AdoMet co-crystal and has no DNA (1HMY [PDB] .pdb), green is with a cytosine target base (3MHT [PDB] .pdb), yellow is with an adenine target base (7MHT [PDB] .pdb), pink is with uracil as the target base (8MHT [PDB] .pdb), black has an abasic target base (9MHT [PDB] .pdb), and blue is M.HaeIII with a cytosine target base (1DCT [PDB] .pdb). Although the DNA component of the abasic structure (black) is difficult to see due to the superimpositions, it can be seen upon careful inspection.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mutant Design—We used alanine replacements in our study because it is the most common amino acid, is relatively unrestricted in conformation, and has a small and inert side chain (38). The mutated residues are not directly implicated in catalysis and, with the exception of Val121 (4 Å), are distal (13–20 Å) from the active site. Within the region selected, Lys122, Asn123, Phe124, and Ser126 were omitted because of likely DNA interactions, based on inspection of the 3MHT [PDB] co-crystal structure. Ala125 was also omitted because it is already alanine. The selected region presents a stretch of peptide that molecular dynamic simulations have predicted to make anti-correlated motions to regions of peptide on the opposite side of the active site, and these motions are believed to be critical for catalysis (7). Anti-correlated motions involve coupled spatial displacements of two elements toward and away from each other in time.

Purification and Characterization—Purified proteins were initially characterized to confirm that appropriate mutations had been made, which included DNA sequencing of the encoding plasmid, peptide sequencing by mass spectrometry, and circular dichroism. Circular dichroism spectra for WT and mutant M.HhaI proteins all showed a high degree of similarity, and representative data for Val121 -> Ala and WT M.HhaI are shown in Fig. 2. DNA sequencing confirmed no secondary mutations had been made, and mass spectrometry data of M.HhaI peptide fragments confirmed that the alanine mutations were appropriately made. Tryptic digested M.HhaI peptide fragment 115–122 was observed at 483.27 (483.26 theoretical) in all WT samples and was shifted in each mutant spectrum because of the alanine mutation altering the molecular mass of the fragment. The Val121 -> Ala fragment was observed at 469.31 (469.24 theoretical) and is representative of other mutant proteins. The mass spectrometric analysis also revealed that Asn129 is partially deaminated to Asp129 in all M.HhaI mutants and WT (data not shown). Inspection of peak clusters from MS/MS data of a peptide fragment (residues 123–137) showed heterogeneity in all peaks for amino acids130–137 and not for amino acids 123–129. In addition, MS/MS peak values revealed the contaminant peptide fragment had a mass 1 unit higher than the WT fragment that disappeared when Asn129 was removed. This mass difference is accountable only by an asparagine that was deamidated to an aspartic acid (39). Although not previously reported for M.HhaI, such post-translational modifications are well characterized and can potentially affect the function of an enzyme (40). We believe the deamidations at position 129 are unlikely to alter the function of the WT or mutant M.HhaI proteins. The Asn129 -> Ala mutant has functional parameters that are indistinguishable from the WT protein. The other M.HhaI mutants have similar levels of Asn129 deamidation yet, other than the Val121 -> Ala mutant, have WT functional parameters. Finally, Asn129 is a considerable distance from the active site. In summary, it is highly unlikely that the large effect on DNA binding observed for the Val121 -> Ala mutant is derived from the double mutation as opposed to just the valine substitution.



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  		FIG. 2.
Circular diochroism spectra were collected for WT M.HhaI (dotted line) and the Val121 -> Ala mutant (solid line). The spectra of alanine mutants 127–132 are also similar to WT.

 
AdoMet Equilibrium Dissociation Constant—M.HhaI has a single tryptophan residue (Trp41) located in the AdoMet binding pocket; AdoMet binding results in fluorescence quenching. Steady state native protein fluorescence can be used to determine KDAdoMet, which provides a direct measure of the functional integrity and affinity of the mutant for AdoMet (9). Upon addition of AdoMet, a sharp decrease in native protein fluorescence of M.HhaI is seen with an emission maximum blue shift, further confirming that Trp41 is being shielded from the aqueous solvent by AdoMet. AdoMet titrations provide fluorescence data that yield KDAdoMet values for mutant and WT proteins (see Table I and Fig. 3). Since all the mutants show near WT values, including Val121 -> Ala, we conclude that the AdoMet binding pocket, an intimate component of the active site, is structurally unperturbed.


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  		TABLE I
Thermodynamic constants for WT and mutant M.HhaI

KDDNA-Abox for Val121-> Ala M.HhaI is ~105-fold down versus WT, whereas KDAdoMet and KDABMbox-DNA have near WT values. KDAdoMet values reflect affinities for the AdoMet substrate, whereas KDDNA values reflect affinities for two different DNA substrates, Abox DNA (unmethylated) and ABMbox DNA (abasic-hemimethylated). Errors were calculated using standard deviation analysis.

 



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  		FIG. 3.
AdoMet affinity determination by native protein fluorescence of M.HhaI with AdoMet titrations at room temperature. WT data are shown as filled squares, the Val121 -> Ala mutant as open circles, and Asn129 -> Ala mutant as open diamonds. Error bars are shown.

 
Steady State Assays—Catalytic turnover constants (kcat) were obtained by a burst assay and the data fit to a linear equation. Fig. 4 shows burst data for Val121 -> Ala, Gly130 -> Ala, and WT enzymes. The burst magnitude for each assay obtained by extrapolating to zero time was used to determine the concentration of active WT enzyme used in the assay (37). The burst is caused by the fast processes leading up to and including DNA methylation (kmethyl transfer) followed by a slow and final step of DNA release, which causes the amount of initial product formed at time 0 to appear greater than 0 (9). kcat values were obtained from the linear portion of the product versus time profile. The kcat values for all mutants other than Val121 -> Ala were similar and close to values published previously for the WT enzyme (0.14 ± 0.02 (s–1) (see Table II)) (9). Val121 -> Ala showed no detectable activity in this assay (see Fig. 4).



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  		FIG. 4.
Burst product formation over 60 s with 1.2 µM AdoMet and 1 µM Abox DNA in MR buffer incubated at 37 °C. Enzyme addition initiated the reaction. Concentrations were 10 nM ({square}, {triangleup}, and {circ}) and 20 nM ({blacksquare}, {blacktriangleup}, and ) with WT M.HhaI ({square}), Val121 -> Ala ({circ}), and Gly130 -> Ala ({triangleup}). Time points were taken at 10-s intervals. Both symbols for Val121 -> Ala ({circ}, ) are shown but overlap. Error bars are shown.

 


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  		TABLE II
Kinetic analysis of WT and mutant M.HhaI enzymes

kcat for Val121-> Ala is ~105-fold down versus WT, and kmethylation and kinactivation values were not detectable using this assay. His127-> Ala also shows a 10-fold decrease in kinactivation from WT values. kmethylation is the methyl transfer rate constant measured with Abox DNA (unmethylated); kinactivation is the inactivation constant measured with FAMbox DNA (hemimethylated), and kcat is the catalytic turnover constant. Errors were calculated using standard deviation analysis.

 
We used an independent measure of kcat to assess the Val121 -> Ala mutant (see Fig. 5). This assay uses much higher concentrations of mutant enzyme and requires multiple turnovers. kcat is ~105-fold lower for Val121 -> Ala than WT based on this assay. This drastic change of kcat reveals that Val121-> Ala can catalyze methylation but is much worse than the WT enzyme.



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  		FIG. 5.
Steady state product formation over 60 min with 125 nM AdoMet and 100 nM Abox DNA for WT and 10 µM AdoMet and 10 µM Abox DNA for the Val121 -> Ala mutant in PD buffer incubated at 37 °C. Enzyme addition initiated the reaction and enzyme concentrations were 8 µM for Val121 -> Ala ({square}) and 100 pM for WT ({circ}). Error bars are shown.

 
Single Turnover Assays—Single turnover experiments probe specific catalytic steps of M.HhaI that are not revealed by steady state kinetics (9, 34). We used Abox and FAMbox DNA substrates to determine kmethylation and kinactivation values, respectively. The observed methylation of the Abox DNA substrate formally includes all steps through kmethylation (see Fig. 6 and Fig. 7, Equation A). Recent work from our group2 has indicated that kmethylation is the rate-limiting step under single turnover conditions. Thus, kmethylation determined under single turnover conditions with Abox DNA substrates reflects the transition involving actual methyl transfer. The single turnover constant measured with FAMbox DNA is also dominated by methyl transfer kinetics, as predicted previously (41). FAMbox DNA inactivates M.HhaI by preventing the {beta}-elimination step, and the enzyme is unable to remove the C5 fluorine atom (11). This produces a stable covalent complex between Cys81 of the enzyme and the C6 carbon of the target base (see Fig. 7, Equation B). kinactivation was shown previously to be ~400-fold slower than kmethylation (10), and we find a similar relationship here (see Fig. 8 and Table II). Since the catalytic process is slowed down so dramatically, kinetic studies with the mechanism-based inhibitor, FAMbox, provide a more sensitive measure of how M.HhaI mutants are impacted in catalysis. This is seen in Table II and Fig. 8 which show that the His127 -> Ala mutant is ~10-fold slower for the FAMbox DNA substrate and has near WT rates for the Abox DNA substrate.



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  		FIG. 6.
Single turnover product formation over 60 s with 3 µM AdoMet and 100 nM Abox DNA in MR buffer incubated at 6 °C. Enzyme addition to 0.4 µM (3.0 µM for Val121 -> Ala) initiated the reaction, and time points were taken at 4, 8, 12, 16, 25, 40, and 60 s. WT is shown as filled squares, the Val121 -> Ala mutant as open circles, and the Asn129 -> Ala mutant as open triangles. Error bars are shown.

 



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  		FIG. 7.
Chemical mechanisms of M.HhaI are shown for the WT reaction with a cognate target base, Abox DNA, and for the 5-fluorocytosine as the target base, FAMbox DNA. Equations A and B show kinetic steps of M.H-haI that correlate to the chemical steps shown below the equations. These rates were examined in this study. These equations are an approximation because several steps are in fact reversible, which is not reflected in these equations.

 



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  		FIG. 8.
Single turnover product formation over 120 min with 800 nM AdoMet and 750 nM enzyme in PD buffer at 37 °C. Reactions were initiated by FAMbox DNA addition to 300 nM, and time points were taken at 2, 4, 10, 30, 70, and 120 min. WT ({blacksquare}), Val121 -> Ala ({circ}), and His127 -> Ala ({triangleup}) are shown. Error bars are shown.

 
DNA Equilibrium Dissociation Constants—DNA dissociation constants (KDDNA) were determined by gel mobility shift assay for multiple DNA substrates (see Table I and Fig. 9). All mutants except Val121 -> Ala show near WT dissociation constants with the Abox DNA substrate (KDDNA-Abox). In order to quantify the impact the Val121 -> Ala mutation had on DNA binding of the Abox DNA substrate, a large excess of enzyme (5 µM) was used. Densitometry of bands of shifted enzyme-DNA complexes within gels between WT and the Val121 -> Ala mutant at high concentrations revealed at least a 105-fold loss in DNA affinity; this is shown in the KDDNA-Abox values seen in Table I and Fig. 9.



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  		FIG. 9.
DNA affinity determination by gel mobility analysis. PAGE with WT and the Val121 -> Ala mutant M.HhaI and two different DNA substrates, one with a cognate target base, Abox DNA, and a second with an abasic target base, ABMbox DNA. Enzyme concentrations are shown in each lane. KDDNA values obtained from this analysis are reported in Table I.

 
WT M.HhaI binds mismatch, abasic, and nicked DNA substrates with tight affinity (13); this is confirmed in the KDDNA values reported for ABMbox DNA versus Abox DNA by the WT enzyme (see Table I and Fig. 9). Most interesting, the Val121 -> Ala gel shift with ABMbox DNA generated a dissociation constant (KDABMbox-DNA) near WT values. Thus, removal of the target cytosine base results in at least a 105 increase in affinity of the mutant for its DNA substrate, suggesting that Val121 plays a critical role in extrahelical base stabilization.

Superimpositions—Root mean square deviation values were calculated for each superimposition shown in Fig. 10C, and the values all show a small deviation (±0.079 Å) from the average value of 0.292 Å. Root mean square deviation values reflect the similarity in the structures seen in Fig. 10C, which also reveals how the two co-crystallized cytosine methyltransferases, M.HhaI and M.HaeIII, have almost identical active sites (see green and blue structures, respectively, in Fig. 10C). Superimpositions of the M.HhaI structures show that Arg165 occupies a position distal from the 5'-phosphate for the binary structure with AdoMet and the tertiary structure with an abasic target site (see red and black structures, respectively, in Fig. 10C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Dynamics studies on M.HhaI show that the extrahelical cytosine within the active site of the enzyme has very low B-factors (7). This rigidity is in part because of restricted {chi} angles of the target base which is critical for the nucleophilic attack by Cys81, transfer of the methyl group from the AdoMet cofactor, and hydrogen bonding interactions with Arg165 and Glu119 (see Fig. 10, A and B). Val121 and other nearby residues appear to be important for active site compression and stabilization of the extrahelical cytosine by determining the appropriate {chi} angle (7). We sought to investigate the importance of Val121 and the adjacent peptide element (residues 127–132) on the active site compression and extrahelical cytosine positioning (7, 19). The adjacent peptide element includes an unusual {alpha}{beta}-turn with a hydrogen bond between His127 and Thr132 (see Fig. 1C) which could help stabilize and maintain active site interactions involving Val121.

Tables I and II summarize our structural and functional analyses of Val121 -> Ala and six additional M.HhaI mutants, all involving alanine substitutions. All mutants have near WT secondary structure, as determined by circular dichroism (see Fig. 2). All mutants except for Val121 -> Ala have near WT values for kcat and kmethylation, whereas kinactivation shows an ~10-fold decrease for the His127 -> Ala mutant and no detectable signal for the Val121 -> Ala mutant (see Table II). All mutants have near WT affinity for AdoMet and, except for Val121 -> Ala, have near WT affinity for Abox DNA (see Table I), also supporting the idea that these alanine replacements left the tertiary protein structures unperturbed. The Val121 -> Ala mutant was dramatically altered in both DNA binding and catalysis, showing losses in activity comparable to the glycine mutant of the nucleophilic Cys81 in M.HhaI (16) and the alanine mutant of Cys186 in EcoRII (42).

Our mutant analysis probed the importance of the putative hydrogen bond between His127 and Thr132 in M.HhaI that was suspected to be necessary for positioning Val121 above the extrahelical cytosine (see Fig. 1C). Neither His127-> Ala nor Thr132 -> Ala shows a significant change in kcat or the presteady state parameter, kmethylation, measured with the Abox DNA substrate (see Table II). Only in the case of kinactivation, measured with the mechanism-based inhibitor (5-fluorocytosine at the target base, FAMbox DNA), is the His127 -> Ala mutant ~10-fold slower than the WT enzyme (see Fig. 8 and Table II). FAMbox DNA decreases the observed rates for M.H-haI methylation by ~400-fold (see Fig. 7, Equation B) (10). Thus, only when the energetics of the methylation reaction are made significantly more difficult do we detect subtle differences in specific catalytic steps for mutants relative to WT M.HhaI. In sum, the alanine substitutions distal from the active site (residues 127–132) appear to have minimal impacts on catalysis. We found this somewhat surprising given the importance of Val121 to both DNA binding and catalysis (see below).

The Val121 -> Ala mutant showed no detectable activity in the single turnover experiments with either Abox or FAMbox DNA, and an ~105 lower steady state turnover rate (kcat) than WT M.HhaI (see Figs. 5, 6, and 8 and Table II). Inspection of several M.HhaI-DNA co-crystal structures suggested that Val121 may be important for maintaining protein interactions with the target base. Moreover, we hypothesized that the Val121 -> Ala mutant might preclude such interactions and that removal of the target base could recover some DNA binding because such putative negative interactions would be removed. Surprisingly, the mutant binds abasic DNA lacking the target cytosine base (ABMbox DNA) with near WT affinity (12); hence, the removal of this cytosine base recovers at least 105-fold binding energy (see Table I and Fig. 9). Whereas a definitive mechanistic understanding will require a co-crystal structure of the mutant enzyme-DNA complex, these results have direct bearing on the induced fit mechanisms of DNA binding, base flipping, and loop motions in M.HhaI.

An interesting conclusion from our kinetic and thermodynamic data is that prevention of the complete base flipping process is detrimental to DNA binding. Furthermore, this loss of DNA affinity can be accomplished by perturbing interactions with the base of the target nucleotide, even though the WT enzyme is highly accommodating for mismatched nucleotides. One obvious interaction previously suggested to contribute to the base flipping process involves protein-phosphate interactions (8, 27, 31, 43–45); for M.HhaI, this includes the phosphate 5' to the flipped cytosine and Arg165. Below we discuss this in detail in the context of a conserved motif, but we propose that any perturbation in this interaction is unlikely to contribute significantly to the mutant's loss in cognate DNA binding affinity. The tertiary co-crystal structure of WT M.HhaI with an abasic DNA substrate and AdoHcy (black structure, Fig. 10C; 9MHT [PDB] ) (21) and the binary M.HhaI-AdoMet co-crystal structure (red structure, Fig. 10C; 3MHT [PDB] ) (20) shows that Arg165 moves away from the DNA phosphate and into an active site cavity left vacant by the missing base. Arg165 moves 2.3 Å away from the 5'-phosphate between the co-crystal structure of the cognate cytosine target site (3MHT [PDB] ) and the abasic target site (9MHT [PDB] ), and this net distance (5.3 Å) is too great to allow any hydrogen bonding between Arg165 and the 5'-phosphate (see Fig. 10C). Thus, Arg165 does not appear to make critical interactions to stabilize the 5'-phosphate with an abasic DNA substrate, which has a tight affinity (see Table I). Furthermore, whatever interactions are critical for the tight binding of abasic DNA are retained in the Val121 -> Ala mutant because it shows nearly the same affinity for this site as the WT enzyme.

The dramatic loss of cognate DNA binding affinity by the Val121 -> Ala mutant may derive partially from the disruption of direct interactions between Val121 and the extrahelical base. Although our motif identification (discussed below), the MD simulations (6, 7), and NMR studies (31) lend some credence to this hypothesis, it seems unlikely that direct interactions between an active site hydrophobic residue and the extrahelical base would account for an ~105 loss of DNA binding when perturbed (46). Furthermore, the fact that WT M.HhaI binds abasic and mismatched DNA with tight affinity argues against the quantitative significance of such interactions (12). A related explanation is that Val121 may be required for the correct assembly of other active site residues, such as Glu119, Arg165, and Cys81, and improper assembly leads to detrimental interactions with the target base (see Fig. 10, A and B). Disruption of critical interactions involving the cytosine base with Glu119, Arg165, and Cys81 could account for the loss in DNA affinity. The ~105 decrease in cognate DNA binding affinity (~7 kcal) corresponds to a loss of two or three hydrogen bonds (46), which could reasonably be assigned to Glu119, Arg165, and the thioether bond made by Cys81 (Fig. 10A) (19). The covalent enzyme-DNA adduct involving Cys81 could also contribute significant binding energy.2 The disruption of these interactions could reasonably reposition residues within the active site in the region normally occupied by the extrahelical cytosine base. The fundamental problems with these explanations are that they fail to account for the lack of tight binding of the cognate DNA site by the Val121 -> Ala mutant and the tight binding of abasic and mismatched sites by the WT M.HhaI.

We therefore propose a mechanism in which tight DNA binding and base flipping are coupled. This induced fit mechanism involves utilization of the binding energy provided by the transition of the enzyme from the initial complex with nonspecific DNA to that involving the cognate site to drive both base flipping and protein loop movement. The most compelling evidence in support of this mechanism at least contributing to our observations with the Val121 -> Ala mutant is that removal of the target cytosine allows the mutant to bind DNA with tight affinity, presumably because base flipping or base stabilization and DNA binding have been uncoupled. Other data support such a coupling, albeit from a different perspective. The WT enzyme binds mismatched and abasic substrates as tightly as the cognate site, despite the loss of specific interactions between the extrahelical base and the active site (13). This observation is somewhat surprising because the extrahelical base is not as well accommodated within the active site as the cognate cytosine. However, the equilibrium between the stacked and flipped state of DNA bases, which is typically ~105 in favor of the stacked state (47), is significantly disturbed with mismatched bases and abasic DNA (48). Thus, the binding preference of M.HhaI for mismatched DNA most likely derives extensively from the increase in concentration of this normally unstable extrahelical conformation. This supports the coupling of tight binding and base flipping because lowering the energy to flip a base increases the available binding energy.

M.HhaI appears to utilize an induced fit mechanism in another context, which we suggest is further coupled to the transitions proposed here. The peptide loop defined by residues 80–99 undergoes a large motion (25Å) upon binding cognate DNA. More precisely, the enzyme-AdoMet complex (20) and the enzyme-DNA-AdoMet complex with nonspecific DNA (22) both show the loop to be positioned in the open conformation. Furthermore, only in the tight ternary complex with specific DNA containing cytosine, abasic, or mismatches at the target base is the loop rearranged in the closed conformation. Most interesting, this loop motion does not appear to require the cofactor, as M.HhaI catalyzes efficient nucleophilic attack and exchange of the C5 hydrogen on the target cytosine in the absence of the cofactor (11).2 This loop was proposed to remain in the open conformation when the furanose ring in the target base is prevented from undergoing sugar pucker transitions thought to be important for base flipping (14). Using abasic DNA molecules with sugar analogs in MD simulations and gel shift experiments, both base flipping and tight DNA binding were shown to be decreased when the sugar torsional angles are restricted (14). Our results and interpretation complement these findings. In both cases, perturbing base flipping impacts DNA affinity, and based on prior structural studies (20, 22), this transition may be coupled to loop movement.

The closed loop makes extensive contacts with the DNA, AdoMet, and most important Val121 (19). We suggest the overall coupling of tight binding, loop motion, and base flipping to be components of an induced fit process, and the Val121 -> Ala mutant has drastically impacted the base flipping component of this process. The two other DNA methyltransferases for which co-crystal structures are available (M.HaeIII and M.TaqI) show very similar loop motions and are thought to share an induced fit/loop motion mechanism (49, 50). The final tightly bound complex is only achievable following loop motion, which requires stabilization of the extrahelical base and/or the correct positioning of active site residues that are normally involved in that stabilization (14). The structural mechanism of how Val121 contributes to the correct assembly of the active site or catalytic loop is being investigated through the study of the Val121 -> Ala M.HhaI-AdoMet-DNA (abasic) co-crystal structure.3

Evidence for the widespread presence and evolutionarily ancient origins of enzymes that stabilize extrahelical bases, including both DNA methyltransferases and repair enzymes, was described previously (8). Considering the importance of Val121 in M.HhaI, we hypothesized that it may form part of a motif involved in base flipping and stabilization. The structures shown in Fig. 11 provide evidence for such a motif involving hydrophobic and charged amino acid residues positioned adjacent to both the extrahelical target base and a proximal phosphate. For M.HhaI, the Val121 side chain is 4.1 Å from the flipped cytosine, 3.5 Å from the 5'-phosphate oxygen of the extrahelical cytosine (O-2P), and 3.7Å from the central guanidinium carbon of Arg165, whereas one nitrogen from the same guanidinium is 3.0Å from the other 5'-phosphate oxygen (O-1P) (see Figs. 10A and 11A). This motif of a positively charged side chain (Arg, Lys, or His), a proximal hydrophobic residue (Ala, Val, Leu, Ile, Pro, or Phe) positioned above the extrahelical base, and adjacent to either 5'- or 3'-phosphate appears in all three methyltransferase co-crystal structures and five of eight DNA repair co-crystal structures (see Fig. 11) that we examined (19, 45, 49–54). Fig. 11D shows the polar Gln144 side chain in place of a positively charged residue in our motif. Although positively charged side chains appear frequently at protein-nucleic acid interfaces, the conserved placement in bacterial and human enzymes of hydrophobic and charged residues adjacent to extrahelical bases and phosphates is compelling. Interestingly, the motif includes interactions with phosphates that are 5' and 3' to the target base, implying that no unique interaction to either phosphate is critical to the function of this motif. Although the mechanism whereby the motif described here functions in either base flipping or stabilization awaits further experiments, it provides a structural signature that supports the previous evolutionary hypothesis (8).



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  		FIG. 11.
Six active site structures of base flipping enzymes are shown. All these enzymes have a positive side chain positioned adjacent to a 5'-phosphate, and the target base in addition to a hydrophobic residue and distances are shown. The conservation of positioning for these enzyme active sites argues that this is an important motif used by base flipping enzymes to facilitate catalysis, and quite possibly base flipping. A is M.HhaI from 3MHT [PDB] .pdb; B is M.TaqI from 1G38 [PDB] .pdb; C is Escherichia coli 3-methyladenine DNA glycosylase from 1DIZ [PDB] .pdb; D is human uracil-DNA glycosylase from 1EMH [PDB] .pdb; E is human 3-methyladenine DNA glycosylase from 1BNK [PDB] .pdb; and F is human 8-oxoguanine glycosylase from 1EBM [PDB] .pdb.

 

   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM 463333 and National Science Foundation Grant MCB-9983125 (to N. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel:. 805-893-8368; Fax: 805-893-4120; E-mail: reich{at}chem.ucsb.edu.

1 The abbreviations used are: M.HhaI, C5 cytosine methyltransferase type I from Haemophilius haemolyticus; WT, wild type; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; MD, molecular dynamics; DTT, dithiothreitol; BSA, bovine serum albumin; MS/MS, tandem mass spectrometry. Back

2 N. Reich and Z. Svedruzic, submitted for publication. Back

3 F. Shieh, R. A. Estabrook, J. Perona, and N. Reich, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. John Perona for structural insights and article review, Dr. Tom Bruice and Dr. Jia Luo for discussions related to MD analysis of M.HhaI, Miguel del Rios and Dr. Blake Gillespie for CD assistance, Dr. James Pavlovich for mass spectrometry assistance, and Dr. Vyas Sharma for technical and kinetic insights.



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