From the
Department of Pharmacology, University of
Colorado Health Sciences Center, Denver Colorado 80262 and the
Department of Biochemistry and College of
Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois
61801
Received for publication, April 10, 2003 , and in revised form, May 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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Type II DNA MTases share a conserved catalytic core structure
(5), composed of a
seven--stranded sheet flanked by three
-helices on each side,
that contains the ligand-binding pockets and active site
(6,
7). The amino acid sequences of
the MTases are not as well conserved as their structures, but they share nine
conserved sequence motifs (7).
On the basis of these motifs, the MTases are subdivided into C5 MTases, which
methylate cytosine at C5, and
,
, and
subclasses of amino
MTases, which methylate either adenine at the N6 position (N6A) or cytosine at
the N4 position (N4C) (7). Of
the conserved motifs, the catalytic motif IV, which contains a conserved PC
for the C5 MTases (8) and a
conserved (D/N/S)PP(Y/F) for the amino MTases
(7), is implicated in catalysis
(9).
RsrI MTase (M.RsrI) is a -class N6A enzyme that recognizes
the palindromic duplex DNA sequence GAATTC and methylates the internal adenine
on each strand (10,
11). It is an isoenzyme of
EcoRI MTase, a
-class MTase, with which it shares very little
sequence homology (16% identity
(10)). M.RsrI is most
homologous in structure and sequence identity (28%) to the N4C MTase PvuII
(6). Previous work partially
characterized kinetic and binding properties of M.RsrI with AdoMet, as well as
the product of the methylation reaction
S-adenosyl-L-homocysteine (AdoHcy) and the specific
inhibitor sinefungin (12). The
structure of M.RsrI revealed a breakdown product of AdoMet,
5'-methylthioadenosine (5'-MTA)
(13) in the ligand-binding
site (14). Here we present the
structures of RsrI MTase bound to the ligands AdoMet, AdoHcy, or
sinefungin. The structures illustrate the similarities and key differences in
ligand binding and explain some observed biochemical properties. We also
present the structure of the catalytically impaired L72P mutant of M.RsrI
(15), which is the first DNA
MTase structure to be determined without bound ligand. This structure offers a
unique opportunity to examine directly the structural changes of DNA MTases
that occur upon ligand binding.
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EXPERIMENTAL PROCEDURES |
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Co-crystallization and Crystal SoakingThe M.RsrI and M.RsrI-L72P proteins were expressed from pET28a+::rsrIM (14) or the mutant rsrIM gene, respectively, and purified as described (12, 14). AdoMet was obtained from New England Biolabs, AdoHcy was obtained from Sigma, and sinefungin was a gift from Margaret Neidenthal (Eli Lilly Laboratories). All three were used without further purification, and concentrations were determined using an extinction coefficient of 15,600 M-1cm-1 at 260 nm. M.RsrI (2.0 mg/ml) was co-crystallized by the addition of AdoMet, AdoHcy, or sinefungin at final concentrations between 1 and 20 mM in a crystallization buffer of 100 mM HEPES, pH = 7.4, 1.5 M Li2SO4. Co-crystals appeared under the same crystallization conditions as did the native protein and had nearly identical morphology (chunky plates of dimensions 400 x 400 x 50 µm) (14). Co-crystals of the M.RsrI-AdoHcy complex were soaked for 6 h in the well solution described above plus 450 mM KBr to produce bromide-substituted crystals for anomalous scattering experiments. M.RsrI-L72P crystals were crystallized using the same conditions but without the addition of ligand, and this resulted in crystals of similar morphology.
Data Collection and Structure DeterminationA single crystal of each complex was used to collect diffraction data using Molecular Structure Corporation R-Axis IV and R-Axis IV++ detectors in-house and at Rigaku-MSC (The Woodlands, TX). The L72P data and anomalous diffraction data for the bromide-soaked M.RsrI-AdoHcy complex at a wavelength of 0.9184 Å were collected on beamlines 19-ID and 19-BM (Advanced Photon Source at Argonne National Laboratory), respectively. In all cases, cryoprotection was achieved by drawing the crystals through paraffin oil before freezing in liquid nitrogen, and data were collected at -180 °C. The cell dimensions (70.42 x 130.25 x 67.28 Å) of the crystals of the complexes are similar (Table I). Data were processed using the HKL programs HKL2000 (16), Denzo and Scalepack (16), or d*TREK (Rigaku-MSC).
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Rigid body refinement of the M.RsrI model (PDB number 1EG2 [PDB] ) with the individual data sets gave R values of below 30% (Table I). Electron density maps of the active site were inspected for the presence of the ligands. The ligand models and M.RsrI-L72P mutation were built using O (17). The models were refined using the maximum likelihood procedure implemented in CNS and simulated annealing omit maps (18). Refinement of individual B-factors and the addition of water molecules completed the model building. PROCHECK (19) and CNS (18) were used to evaluate the model geometry. To confirm the absence of ligand in the L72P structure, ligand was refined in the model, and the occupancy was determined to be less than 0.5. Anomalous difference Fourier maps were used to locate the bromide ions within the wild-type model.
Structural AnalysisCoordinates of MTase crystal structures were obtained from the Protein Data Bank: M.RsrI, 1EG2 [PDB] (14); M.TaqI-SAM, 2ADM [PDB] (20); M.TaqI-SFG, 1AQJ [PDB] (20); M.TaqI-SAH, 1AQI [PDB] (20); M.PvuII-SAH, 1BOO [PDB] (6); M.HhaI-SAM, 2HMY [PDB] (21); M.DpnII2-SAM, 2DPM [PDB] (22). The root-mean-squared deviations (r.m.s.d.) between structures were determined using the Swiss PDB Viewer (23). The torsion angle analysis was performed using O (17) and Excel (Microsoft). The sugar puckers were determined by inspection. The figures were generated using BOBSCRIPT, MOLSCRIPT (24), Swiss PDB Viewer (23), Raster3D (25), POV-RAY, and PhotoShop (Adobe).
Coordinate DepositionAtomic coordinates of the structures reported were deposited in the Protein Data Bank under the following accession numbers: M.RsrI-AdoMet, 1NW5; M.RsrI-AdoHcy, 1NW7; M.RsrI-sinefungin, 1NW6; M.RsrI-L72P, 1NW8.
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RESULTS |
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In addition to differences in tail orientations of the AdoHcy and sinefungin complexes, the ribose conformations differed from those of AdoMet. Both AdoHcy and sinefungin had C2'-endo sugar puckers when compared with C1'-exo for AdoMet and 5'-MTA. Since all of the ribose atoms formed the same hydrogen bonds (Table III and Fig. 3A), and the only positional differences in the adenine and ribose moieties were the C4' and C5' atoms, which are directly connected to the tail moiety, these differences in sugar conformations appeared to be caused by the differences in the tail orientations. The formation of a hydrogen bond, or lack thereof (AdoHcy), to the charged sulfur (AdoMet) or nitrogen (sinefungin) by Thr225 (Table III) may contribute to the different sugar conformations because these charged atoms occupy different positions in the liganded M.RsrI structures (the nitrogen in sinefungin corresponds to the carbon of the activated methyl group in sinefungin). The adenine rings of AdoMet and sinefungin occupied nearly identical positions, whereas that of AdoHcy was twisted slightly away from the ribose relative to the other two. The nearly identical binding of the nucleoside moiety of the three ligands by M.RsrI contrasts with the ligands bound to M.TaqI, as shown in Fig. 3B, where the ligand nucleoside moiety positions are shifted slightly from one another (20).
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Comparison of the ligands bound to M.RsrI with those bound to other DNA MTases revealed a wide variety of sugar puckers and torsion angles, but the torsion angles of AdoMet are more similar to one another than the torsion angles of the other ligands (Table IV). AdoMet, with the exception of the M.TaqI-AdoMet structure, assumes a sugar pucker of C1'-exo in all of the crystal structures, which may be due to the common bent tail conformation and the required orientation of the methyl being donated. With the exception of M.PvuII, all the AdoMet structures have a bent tail conformation, whereas the AdoHcy and sinefungin structures adopt an extended conformation. In the case of M.PvuII, the bound AdoHcy assumes a bent conformation similar to that of AdoMet. However, AdoHcy appears in the PDB file for the PvuII structure only because of weak methyl group density as the crystals were grown in the presence of AdoMet and the structure was discussed as though AdoMet were bound (6).2 Our analysis of ligand binding orientations supports the assignment of AdoMet as the ligand bound to M.PvuII as AdoMet is the only ligand that binds with a bent tail conformation in all of the other MTase structures.
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In addition to differences in ligand configurations, M.RsrI amino acid side
chains surrounding the ligands changed position and structure
(Fig. 4, A and
B). Table
III lists the ligand-enzyme contacts and shows that the most
noticeable side chain difference is Lys227, which, in the AdoHcy
and sinefungin structures, had shifted into the position occupied previously
by the amino group of the AdoMet tail (Fig.
2). The side chain orientation of Lys227 in the AdoMet
structure is most likely due to repulsion by the -amino group of the
AdoMet tail. M.RsrI in the AdoHcy complex differed (r.m.s.d. for all atoms)
from both the AdoMet- and sinefungin-bound structures at residues
Trp84 and Cys45 by 0.6 and 0.7 Å, respectively.
Both of these amino acids adjoined the adenine ring of the ligand, and their
change in position reflects the slightly different orientation of the adenine
ring in the AdoHcy structure.
Comparison of the AdoMet, AdoHcy, and sinefungin-bound M.RsrI structures to the M.RsrI native structure, which has 5'-methylthioadenosine (5'-MTA structure) in the active site, reveals additional changes in the enzyme structure surrounding the ligand-binding site (14). The differences between the AdoMet structure and 5'-MTA structure only are reported in Table II because all three co-crystal structures differ from the 5'-MTA structure in analogous regions. The largest differences between the two compared structures occurred in the small helix/loop adjacent to the ligand-binding site (residues 214225). This region overlaps the loop that contacts the tail of the bound ligand (residues 223227), and presumably, occupies a different position in the 5'-MTA structure due to the lack of a complete tail on that ligand. The other large differences between the structures (residues 180197, 215219) were in areas of crystal contacts and probably reflect slight packing differences.
Specific Enzyme Conformational Changes Occur with Ligand BindingThe published structures of all DNA MTases contain bound ligand. Therefore, it was interesting to observe that a catalytically compromised mutant, L72P, identified from a challenge-phage screen (15), did not purify and crystallize with bound ligand. This mutant binds DNA site-specifically, but the activity of the enzyme is reduced by at least 65-fold (15). The L72P mutant of RsrI MTase was crystallized, and the structure was solved by molecular replacement. The structure resolved amino acids 36288 and 297315, and the density for the proline mutation was clearly visible. Global analysis of the structure revealed an r.m.s.d. of 0.4 Å for the protein backbone when compared with the M.RsrI-AdoMet complex. The ligand-binding site lacked ligand electron density, and occupancy refinement with added ligand suggested a low overall occupancy of the site. Therefore, the discrete spheres of electron density in the ligand-binding site were modeled as water molecules (Fig. 3C).
In the region surrounding the L72P mutation, the proline substitution
caused a kink in the loop, which led to a compression of the
loop/-strand on one side of the mutation
(Fig. 4C, I)
and a shift of the end of an
-helix on the other side
(Fig. 4C,
II). Although these changes were not large, the close packing of the
loops in this region appears to have led to secondary changes in the
neighboring loops. The compression caused by the proline appears to cause a
shift in the
-strand containing the catalytic DPPY (6568) away
from the ligand-binding site and also a shift of 0.4 Å in the conserved
tyrosine. The proline kink introduced by the L72P mutation also caused the
helix following the L72P mutation to twist away from the ligand-binding site
and down toward the main body of the protein
(Fig. 4C,
II). The largest deviation in the helix was seen in Trp84,
which forms one of the walls of the ligand-binding site. Trp84
moved away from its position in the AdoMet structure by over 0.5 Å
(Fig. 3D), increasing
the size of the ligand-binding site. The loop and helix (108131)
adjacent to the L72P loop (Fig.
4C, III) on the active site side were also
pushed away from the ligand-binding pocket. This region formed several
contacts across the crystallographic dimer interface, and the movement of this
region led to the formation of additional contacts between monomers, including
two hydrogen bonds from Gln109, where the
-carbon has moved
almost 0.6 A away from its position in the AdoMet structure.
By far the most obvious difference between the ligand-bound and the L72P
structures was the ligand-binding loop on the opposite side of the binding
site from the L72P mutation (Fig.
4C, IV). This loop was displaced from the
ligand-binding site by over 0.5 Å, and combined with the
Trp84 movement, resulted in an increase in the size of the
ligand-binding site from 8.3 x 16.0 Å (measured
Trp84Ala272 and
Thr225Asp46) in the AdoMet structure to 8.5
x 17.6 Å in the L72P structure
(Fig. 3D). The loop,
specifically residues 223227, contacted the methionine tail in the
AdoMet structure (Figs. 2 and
3A), indicating that
the lack of ligand was directly responsible for its structural changes.
Interestingly, the residues involved in these contacts comprise a
HXTXKP motif that is conserved among many members of the
-class of amino MTases, but not M.PvuII
(7). M.PvuII and a second
subgroup of
-class MTases contain a HXTXKP motif at
the corresponding position (7).
Replacement of the Lys in M.RsrI (Lys227) with Phe in M.PvuII and
other MTases may reduce the size and charge of the ligand-binding pocket, and
consequently, chloride ion does not bind in the other MTases.
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DISCUSSION |
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The adenine and ribose portions of the ligands in the M.RsrI co-crystal structures bound in nearly identical orientations. This is in contrast to M.TaqI, the only other MTase crystallized with this range of ligands, where the adenine and ribose moieties are shifted relative to one another by up to 0.8 Å. This difference between the two enzymes is likely due to the N terminus of M.TaqI forming part of the ligand-binding site (20). The freedom of the N terminus to move may increase the flexibility in the M.TaqI ligand-binding site.
An explanation for the two different modes of ligand binding by MTases,
including M.RsrI, appears to arise from the difference in the position of the
positive charge on the three ligands. The sulfur of AdoMet and the
-amino group of sinefungin carry a formal positive charge, whereas
AdoHcy is uncharged at the analogous position
(Fig. 1). In both the AdoMet
and sinefungin structures, Thr225, which is conserved in many
-class MTases (7), forms
a hydrogen bond to the positively charged atom. In order for this hydrogen
bond to form, the tail of AdoMet must assume a bent conformation; however, the
difference in position of the charge on the sulfur in AdoMet when compared
with the position of the charged nitrogen in sinefungin
(Fig. 1) causes the ornithine
tail in sinefungin to form the hydrogen bond by rotating slightly around the
C4'-C5'-CD-CG torsion angle to maximize hydrogen bond strength
(Fig. 3A). AdoHcy
lacks a corresponding charge, so its tail is not forced to bend into the
pocket by the formation of a hydrogen bond to the sulfur.
Relationship of Different Ligand Binding Modes to Affinity The different conformations of the ligand tails are not directly reflected in the ligand equilibrium dissociation values (12). The B-factors for AdoMet and AdoHcy were very similar, indicating similar mobility; however, AdoHcy formed three fewer hydrogen bonds to the protein (two with water-mediated bonds) and binds with a Kd of 8.1 µM, which is similar to that of AdoMet (Kd = 6.1 µM) (12). In contrast, sinefungin formed two fewer hydrogen bonds than AdoMet (one fewer if water-mediated hydrogen bonds are considered) and has higher B-factors, indicating more conformational flexibility, which might contribute to its affinity of 4.6 µM by decreasing the entropic binding penalty. This greater degree of motion and separation of sinefungin relative to the nearby tryptophans (Trp84, Trp88, and Trp140 are within 9 Å) may explain the differences in fluorescence quenching observed between sinefungin (46.5%) and AdoMet (54.4%) (12). The slight movement of the adenine ring and Trp84 (Figs. 3A and 4A) in the AdoHcy-bound structure may also be related to the slight change in fluorescence quenching for AdoHcy (49.5%) relative to AdoMet.
The two contrasting modes of ligand binding revealed by the AdoMet-bound versus the sinefungin- and AdoHcy-bound structures raise an interesting mechanistic question. Does the positioning of the tail in the extended position, as observed for the inhibitor sinefungin and the product AdoHcy, inhibit base flipping? Fluorescence studies to probe base flipping, using DNA fragments containing 2-aminopurine at the target base, performed in the presence of sinefungin, show an increase in fluorescence consistent with the 2-aminopurine base being flipped out of the hydrophobic double helix and into the more polar active site (12). Two hypotheses are consistent with this observation. The tail position observed for sinefungin and AdoHcy in the structure might not interfere with base flipping, so that the ligand tail could continue to occupy its observed position, whereas the active site contains the flipped target base. Alternatively, the ligand tail might adopt the same orientation as AdoMet before DNA binds to the enzyme-sinefungin complex. In both cases, we expect the ligand to bind prior to DNA binding based on the burst kinetics experiments performed previously (12). If the second hypothesis is indeed the case, electron density for the second conformation might be expected in the crystal structure if the AdoMet-like conformation were found in an appreciable fraction of the bound ligands. Although we observed no such density, suggesting an alternate ligand conformation in either the sinefungin or AdoHcy structures, we cannot rule out this possibility without a structure of the enzyme bound to both DNA and sinefungin or AdoHcy.
Chloride Inhibition of M.RsrIThe L72P-, sinefungin-, and AdoHcy-containing structures revealed a chloride in the position occupied by the carboxylate group of the methionine tail in the structure with bound AdoMet (Fig. 2). This result raises the question of whether chloride competes with AdoMet during DNA methylation. M.RsrI activity is sensitive to salt, and the enzyme is inactive above 160 mM NaCl (10). The isozyme EcoRI MTase does not show inhibition by high salt levels. NaCl at 50 mM, but not sodium acetate, inhibited M.RsrI in a qualitative methylation assay (data not shown), suggesting that the effect is chloride-specific and not due to the sodium ions. This identification of a chloride in the active site pocket of L72P, AdoHcy, and sinefungin suggests a mechanism for enzyme inhibition by high salt concentrations. However, detailed kinetic studies will be necessary to determine the nature of the inhibition observed since it might also be due to the second chloride that we observed in the crystal structure.
The second chloride ion identified in the structures appears to play a role in stabilizing the orientation of the putative target recognition domain (Fig. 4). The TRD extends from the globular body of the protein, and the chloride is part of a bridging hydrogen-bonding network between residues 136 and 137 in the body of the protein and residue 206 in the TRD. This chloride may also be partially responsible for enzyme inhibition as it could impede the required mobility of the TRD during DNA binding. This would be consistent with the effects of the D173A mutation isolated previously in this region (15). Asp173 is involved in a second hydrogen-bonding network in the TRD, the disruption of which does lead to a loss of DNA binding in a challenge-phage assay (15).
The bent conformation of the tail of AdoMet illuminates the mechanism of the enzyme. DNA amino MTases transfer the methyl group directly from AdoMet to the target base with inversion of configuration of the methyl hydrogen atoms (26). This requires the methyl group be positioned in the enzyme active site near the target base. Furthermore, because the reaction takes place by an SN2 mechanism (26), a positioning of the bond between the methyl and sulfur along the axis of transfer is preferred. Such stereochemical positioning was observed for the bound AdoMet. In addition, the M.TaqI-DNA crystal structure implicated the conserved NPPY (DPPY in M.RsrI) motif in activating the exocyclic amino group of the target base so as to accept the methyl group (27). Specifically, the Asn and the second Pro formed hydrogen bonds to the amino group of the flipped adenine base of the target sequence. In the structure of RsrI MTase co-crystallized with AdoMet, the amino group of the methionine tail and the side chain of Lys227 form ionic interactions with one of the carboxylate oxygens of the putative catalytic Asp. These two ionic interactions could position the catalytic Asp in the correct orientation to hydrogen bond to, and help activate, the exocyclic amino group on the target base. The carboxylate may also serve as the acceptor for the proton abstracted from the adenine during methyl transfer.
Ligand-induced Structural Changes in M.RsrIThe L72P structure provided a first glimpse into the structural changes that take place upon ligand binding and revealed a potential explanation for the increased affinity for specific DNA when the ligand is bound to the enzyme. This phenomenon is relevant because ligand binding by M.RsrI, and other MTases, increases cognate DNA binding affinity (12, 28, 29). The native M.RsrI crystallized without added ligand, but we observed 5'-MTA bound in the active site, presumably due to co-purification with the enzyme (14). We showed that 5'-MTA acted similarly to other cofactors in enhancing site-specific DNA binding in a gel-shift assay.3 Although 5'-MTA lacks an amino acid side chain, the interactions of the adenine and ribose with the enzyme appear to be sufficient to pull most of the ligand-binding loop into position (Fig. 3A). The L72P structure lacked a bound ligand because either the ligand did not co-purify or the enzyme could not crystallize with bound ligand. The latter possibility is most likely as we have been unable to obtain co-crystals of the L72P mutant in the presence of added AdoMet.
The M.RsrI ligand-binding loop (Fig. 4C, IV) is located on the opposite side of the ligand-binding site from the L72P mutation and makes no other contacts in the crystal structure, which sensitizes it to the occupancy of the ligand-binding site. One interpretation of the movement of this loop is that the ligand is responsible for stabilizing the loop in the position observed in the native structure of the wild-type protein, decreasing the mobility of the loop and, potentially, orienting it for the next step in the reaction. The analogous loop, termed Loop I, in the M.TaqI/DNA structure is important in DNA and ligand binding (27). Given the structural changes that occur upon ligand binding, this loop in M.RsrI probably is involved in DNA contacts as well. In mechanisms where AdoMet obligatorily binds first, which is the case for M.EcoRI (30) and is likely the case for M.RsrI (12), the binding of ligand would move the ligand-binding loop and other loops into a position that would facilitate DNA binding. In contrast, M.HhaI, which may have a mechanism with a random order of binding (31) or a mechanism where DNA binds first (32), would be expected to have binding sites for DNA and AdoMet that can act independently of one another since the ligand is not required for binding DNA. However, ligand binding still increases affinity for specific DNA binding in M.HhaI, but this has been explained as an energetic consequence of filling an otherwise empty space in the enzyme (32).
The structure of the L72P mutant suggests that structural changes caused by subtle shifts of the secondary structures on either side of the mutation might weaken ligand binding. The most obvious amino acid residues responsible for influencing the binding of ligand are the DPPY motif and the Trp84 residue, which moved away from the original positions they occupy in the AdoMet structure. This movement enlarged the ligand-binding site, most likely leading to lower ligand affinity and presumably the loss of the ligand during purification. Decreased affinity combined with the slight movement of the DPPY motif could explain the impaired catalytic activity of the L72P mutant. A second explanation for the reduced activity of the L72P mutant comes from the observation that in the crystal structures of M.HhaI or the M.TaqI-DNA complex, the loop equivalent to the loop containing the M.RsrI L72P mutation is involved in DNA binding and, specifically, stabilizing the flipped base (27, 33). The L72P mutation could change the loop flexibility or conformation in such a way as to either trap the flipped base either in a non-productive conformation or reduce the off rate of the DNA and prevent enzyme turnover. Further kinetic studies will be necessary to verify the contributions of these factors to the activity loss.
ConclusionsThe crystal structure of the L72P mutant and those of the wild-type enzyme co-crystallized with the substrate, AdoMet, the product, AdoHcy, and the inhibitor sinefungin offer insights into ligand binding by MTases. Comparisons with other MTases indicated a pattern in ligand-binding orientations. The identification of a chloride in the active site pocket of L72P, AdoHcy, and sinefungin suggested a mechanism for the inhibition of M.RsrI by high salt concentrations. Furthermore, the missing ligand in the L72P structure revealed a first glimpse into the structural changes that take place upon ligand binding.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1-GM59456
(to M. E. A. C.), National Institutes of Health Grant RO1-GM25621 and a grant
from the University of Illinois at Urbana-Champaign Department of Biochemistry
(to R. I. G.), and the a grant from the University of Illinois Research Board
and National Institutes of Health Grant T32 GM07283 (to C. B. T.). 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.
¶ Present address: Dept. of Biochemistry and Molecular Biology, Mayo Clinic,
Scottsdale, AZ 85259.
|| To whom correspondence should be addressed: Dept. of Pharmacology, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver CO 80262. Tel.: 303-315-0427; Fax: 303-315-7097; E-mail: mair.churchill{at}uchsc.edu.
1 The abbreviations used are: MTase, methyltransferase; AdoMet,
S-adenosyl-L-methionine; AdoHcy,
S-adenosyl-L-homocysteine; M.RsrI, RsrI DNA
methyltransferase; PDB, Protein Data Bank; r.m.s.d., root-mean-squared
deviations; 5'-MTA, 5'-methylthioadenosine; TRD, target
recognition domain.
2 X. Cheng, personal communication.
3 C. Thomas, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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