Structures of Liganded and Unliganded RsrI N6-Adenine DNA Methyltransferase
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m303751200
ISSN1083-351X
AutoresC.B. Thomas, R.D. Scavetta, Richard Gumport, Mair E. A. Churchill,
Tópico(s)Parasitic Infections and Diagnostics
ResumoThe structures of RsrI DNA methyltransferase (M.RsrI) bound to the substrate S-adenosyl-l-methionine (AdoMet), the product S-adenosyl-l-homocysteine (AdoHcy), the inhibitor sinefungin, as well as a mutant apo-enzyme have been determined by x-ray crystallography. Two distinct binding configurations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group toward the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. Analysis of ligand binding in comparison with other DNA methyltransferases reveals a small, common subset of available conformations for the ligand. The structures of M.RsrI with the non-substrate ligands contained a bound chloride ion in the AdoMet carboxylate-binding pocket, explaining its inhibition by chloride salts. The L72P mutant of M.RsrI is the first DNA methyltransferase structure without bound ligand. With respect to the wild-type protein, it had a larger ligand-binding pocket and displayed movement of a loop (223–227) that is responsible for binding the ligand, which may account for the weaker affinity of the L72P mutant for AdoMet. These studies show the subtle changes in the tight specific interactions of substrate, product, and an inhibitor with M.RsrI and help explain how each displays its unique effect on the activity of the enzyme. The structures of RsrI DNA methyltransferase (M.RsrI) bound to the substrate S-adenosyl-l-methionine (AdoMet), the product S-adenosyl-l-homocysteine (AdoHcy), the inhibitor sinefungin, as well as a mutant apo-enzyme have been determined by x-ray crystallography. Two distinct binding configurations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group toward the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. Analysis of ligand binding in comparison with other DNA methyltransferases reveals a small, common subset of available conformations for the ligand. The structures of M.RsrI with the non-substrate ligands contained a bound chloride ion in the AdoMet carboxylate-binding pocket, explaining its inhibition by chloride salts. The L72P mutant of M.RsrI is the first DNA methyltransferase structure without bound ligand. With respect to the wild-type protein, it had a larger ligand-binding pocket and displayed movement of a loop (223–227) that is responsible for binding the ligand, which may account for the weaker affinity of the L72P mutant for AdoMet. These studies show the subtle changes in the tight specific interactions of substrate, product, and an inhibitor with M.RsrI and help explain how each displays its unique effect on the activity of the enzyme. Biological DNA methylation is ubiquitous and important in many cellular mechanisms including genetic imprinting, immunity, cancer, and gene regulation (1Paulsen M. Ferguson-Smith A. J. Pathol. 2001; 195: 97-110Crossref PubMed Scopus (218) Google Scholar, 2Low D. Weyand N. Mahan D. Infect. Immun. 2001; 69: 7197-7204Crossref PubMed Scopus (256) Google Scholar, 3Jeltsch A. ChemBioChem. 2002; 3: 274-293Crossref PubMed Google Scholar). In bacteria, DNA methylation protects the host from foreign DNA through the action of restriction modification systems composed of a restriction endonuclease and a DNA modification methyltransferase (MTase). 1The 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. Both enzymes recognize a specific DNA sequence that is usually palindromic. The endonuclease cleaves the DNA unless the MTase has added a methyl group to a base in the recognition sequence. Restriction modification systems are classified by their subunit composition and cofactor requirements (4Bujnicki J.M. Acta Biochim. Pol. 2001; 48: 969-983Crossref PubMed Scopus (9) Google Scholar). The most studied of these classes, type II restriction modification systems, usually have a dimeric endonuclease and a monomeric MTase that requires only the methyl-donating cofactor S-adenosyl-l-methionine (AdoMet) for activity. Type II DNA MTases share a conserved catalytic core structure (5Roberts R.J. Belfort M. Bestor T. Bhagwat A.S. Bickle T.A. Bitinaite J. Blumenthal R.M. Degtyarev S.K. Dryden D.T.F. Dybvig K. Firman K. Gromova E.S. Gumport R.I. Halford S.E. Hattman S. Heitman J. Hornby D.P. Janulaitis A. Jeltsch A. Josephsen J. Kiss A. Klaenhammer T.R. Kobayashi I. Kong H. Kruger D.H. Lacks S. Marinus M.G. Miyahara M. Morgan R.D. Murray N.E. Nagaraja V. Piekarowicz A. Pingoud A. Raleigh E. Rao D.N. Reich N. Repin V.E. Selker E.U. Shaw P.C. Stein D.C. Stoddard B.L. Szybalski W. Trautner T.A. Van Etten J.L. Vitor J.M. Wilson G.G. Xu S.Y. Nucleic Acids Res. 2003; 31: 1805-1812Crossref PubMed Scopus (566) Google Scholar), composed of a seven-β-stranded sheet flanked by three α-helices on each side, that contains the ligand-binding pockets and active site (6Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (170) Google Scholar, 7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar). The amino acid sequences of the MTases are not as well conserved as their structures, but they share nine conserved sequence motifs (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar). On the basis of these motifs, the MTases are subdivided into C5 MTases, which methylate cytosine at C5, and α, β, and γ subclasses of amino MTases, which methylate either adenine at the N6 position (N6A) or cytosine at the N4 position (N4C) (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar). Of the conserved motifs, the catalytic motif IV, which contains a conserved PC for the C5 MTases (8Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar) and a conserved (D/N/S)PP(Y/F) for the amino MTases (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar), is implicated in catalysis (9Schubert H.L. Blumenthal R.M. Cheng X. Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). RsrI MTase (M.RsrI) is a β-class N6A enzyme that recognizes the palindromic duplex DNA sequence GAATTC and methylates the internal adenine on each strand (10Kaszubska W. Aiken C. O'Connor C.D. Gumport R.I. Nucleic Acids Res. 1989; 17: 10403-10425Crossref PubMed Scopus (39) Google Scholar, 11Kaszubska W. Webb H.K. Gumport R.I. Gene (Amst.). 1992; 118: 5-11Crossref PubMed Scopus (23) Google Scholar). It is an isoenzyme of EcoRI MTase, a γ-class MTase, with which it shares very little sequence homology (16% identity (10Kaszubska W. Aiken C. O'Connor C.D. Gumport R.I. Nucleic Acids Res. 1989; 17: 10403-10425Crossref PubMed Scopus (39) Google Scholar)). M.RsrI is most homologous in structure and sequence identity (28%) to the N4C MTase PvuII (6Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (170) Google Scholar). 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 (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: 3962-3971Crossref PubMed Google Scholar). The structure of M.RsrI revealed a breakdown product of AdoMet, 5′-methylthioadenosine (5′-MTA) (13Hoffman J.L. Biochemistry. 1986; 25: 4444-4449Crossref PubMed Scopus (165) Google Scholar) in the ligand-binding site (14Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar). 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 (15Szegedi S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: 3972-3981Crossref PubMed Google Scholar), 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. Generating the L72P Histidine-tagged Mutant—The M.RsrI-L72P mutation was originally isolated in a genetic screen (15Szegedi S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: 3972-3981Crossref PubMed Google Scholar). The histidine-tagged construct used in the initial structure study was mutated by previously described methods (14Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar, 15Szegedi S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: 3972-3981Crossref PubMed Google Scholar). Briefly, whole plasmid PCR with mutagenic primers containing the desired mutation was used to introduce the L72P mutation into the plasmid DNA. The mutation also introduced an R.NgoMIV site, which was used to screen for plasmids containing the desired mutation. The presence of the desired mutation and the absence of unintended changes were verified by DNA sequencing. Co-crystallization and Crystal Soaking—The M.RsrI and M.RsrI-L72P proteins were expressed from pET28a+::rsrIM (14Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar) or the mutant rsrIM gene, respectively, and purified as described (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: 3962-3971Crossref PubMed Google Scholar, 14Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar). 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 × 400 × 50 μm) (14Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar). 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 Determination—A 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 × 130.25 × 67.28 Å) of the crystals of the complexes are similar (Table I). Data were processed using the HKL programs HKL2000 (16Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38782) Google Scholar), Denzo and Scalepack (16Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38782) Google Scholar), or d*TREK (Rigaku-MSC).Table IStatistics of data collection and refinementModelAdoMetAdoHcySinefunginAdoHcy + KBrL72PCell dimensions (Å)70.36, 129.06, 67.1171.17, 130.75, 67.5270.43, 130.07, 67.2271.27, 131.68, 67.4771.6, 131.38, 67.54Wavelength (Å)1.54181.54181.54180.9184Resolution range (Å)20-2.05 (2.09-2.05)35-2.10 (2.18-2.10)28-1.94 (2.01-1.94)20-2.40 (2.49-2.40)50-2.25 (2.33-2.25)Completeness (%)93.2 (70.7)99.1 (100.0)99.8 (99.8)96.4 (97.7)90.9 (85.5)R symaR sym = ΣI-〈I〉/ΣI0.028 (0.295)0.080 (0.163)0.038 (0.167)0.047 (0.240)0.055 (0.119)〈I/(σ)〉16.3 (2.3)7.5 (2.7)22.5 (6.5)24.7 (4.9)Observed reflections43,60754,426 (5886)78,952 (7600)23,171 (2305)Unique reflections18,235 (681)18,591 (1844)23,268 (2292)12,282 (1203)13,692 (1146)R-valuebR-factor and R free (34) are calculated for 90 and 10% of the data, respectively0.20 (0.24)0.21 (0.25)0.22 (0.25)NRcNR, not refined; the data set was only used for electron density maps0.20 (0.24)Free R-valuebR-factor and R free (34) are calculated for 90 and 10% of the data, respectively0.24 (0.28)0.25 (0.30)0.25 (0.31)0.24 (0.27)Non-hydrogen protein atoms2146215521552158Non-hydrogen ligand atoms2726270Waters144155136148Ions1222Average B-factor (Å2)29.126.427.827.3r.m.s. deviation from idealityNRBond lengths (Å)0.0060.0060.0070.007Bond angles (°)1.151.151.201.20Dihedral angles (°)22.923.022.622.9Impropers (°)0.790.780.800.78a R sym = ΣI-〈I〉/ΣIb R-factor and R free (34Brünger A.T. Methods Enzymol. 1997; 277: 366-396Crossref PubMed Scopus (279) Google Scholar) are calculated for 90 and 10% of the data, respectivelyc NR, not refined; the data set was only used for electron density maps Open table in a new tab Rigid body refinement of the M.RsrI model (PDB number 1EG2) 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 (17Jones T.A. Zou J.Y. Cowan S.W. Kjelgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13055) Google Scholar). The models were refined using the maximum likelihood procedure implemented in CNS and simulated annealing omit maps (18Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17025) Google Scholar). Refinement of individual B-factors and the addition of water molecules completed the model building. PROCHECK (19Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Crossref Google Scholar) and CNS (18Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17025) Google Scholar) 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 Analysis—Coordinates of MTase crystal structures were obtained from the Protein Data Bank: M.RsrI, 1EG2 (14Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar); M.TaqI-SAM, 2ADM (20Schluckebier G. Kozak M. Bleimling N. Weinhold E. Saenger W. J. Mol. Biol. 1997; 265: 56-67Crossref PubMed Scopus (111) Google Scholar); M.TaqI-SFG, 1AQJ (20Schluckebier G. Kozak M. Bleimling N. Weinhold E. Saenger W. J. Mol. Biol. 1997; 265: 56-67Crossref PubMed Scopus (111) Google Scholar); M.TaqI-SAH, 1AQI (20Schluckebier G. Kozak M. Bleimling N. Weinhold E. Saenger W. J. Mol. Biol. 1997; 265: 56-67Crossref PubMed Scopus (111) Google Scholar); M.PvuII-SAH, 1BOO (6Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (170) Google Scholar); M.HhaI-SAM, 2HMY (21O'Gara M. Zhang X. Roberts R.J. Cheng X. J. Mol. Biol. 1999; 287: 201-209Crossref PubMed Scopus (66) Google Scholar); M.DpnII2-SAM, 2DPM (22Tran P.H. Korszun Z.R. Cerritelli S. Springhorn S.S. Lacks S.A. Structure. 1998; 6: 1563-1575Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The root-mean-squared deviations (r.m.s.d.) between structures were determined using the Swiss PDB Viewer (23Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9795) Google Scholar). The torsion angle analysis was performed using O (17Jones T.A. Zou J.Y. Cowan S.W. Kjelgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13055) Google Scholar) and Excel (Microsoft). The sugar puckers were determined by inspection. The figures were generated using BOBSCRIPT, MOLSCRIPT (24Kraulis P.J. J. Appl. Cryst. 1991; 24: 946-950Crossref Google Scholar), Swiss PDB Viewer (23Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9795) Google Scholar), Raster3D (25Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3880) Google Scholar), POV-RAY, and PhotoShop (Adobe). Coordinate Deposition—Atomic 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. To understand better the structure-function relationships of RsrI MTase, we co-crystallized M.RsrI with the substrate AdoMet, the product AdoHcy, or the inhibitor sinefungin. The crystals diffracted beyond 2.3 Å, and the structures were solved directly by molecular replacement using the previously determined native structure (14Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar). Because no electron density for the N-terminal 35 amino acids and the C-terminal 5 amino acids was observed, the final models included amino acids 36–287 and 297–314 for the enzyme-AdoMet complex and amino acids 36–288 and 297–314 for the AdoHcy or sinefungin complexes. The electron density for each ligand was well resolved (see Fig. 2). Overall, the protein structures are similar to each other and to the native structure as indicated by the small r.m.s.d. of the backbone and side chain atoms (Table II). However, distinct differences were observed between the bound ligand structures when compared with either each other or to the previously reported structure of M.RsrI bound to the AdoMet degradation product 5′-MTA (native structure) (14Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar).Table IIStructural differences between native and ligand-bound structuresStructures comparedBackbone r.m.s.d.Side chain r.m.s.d.Overall r.m.s.d.Residues (Cα r.m.s.d. > 0.3 Å)ÅAdoMet-Native0.170.710.51190-197, 214-225AdoHcy-Native0.200.980.7071-79, 180-183, 191-194Sinefungin-Native0.150.670.48214-224AdoHcy-AdoMet0.251.030.7471-74, 84-86, 180-198, 215-219Sinefungin-AdoMet0.160.780.56191-196, 215-217L72P-AdoMet0.391.170.8771-79, 84-89, 108-114, 180-198, 211-231, 276-287 Open table in a new tab Ligands Adopt Distinct Conformations in Binding to M.RsrI—The obvious difference among the three M.RsrI-ligand structures was the orientation of the non-nucleoside, or "tail," portion of the bound ligand (see Figs. 2 and 3A). In the AdoMet complex, the methionine tail had a C4′-C5′-SD-CG torsion angle (Fig. 1) of -70°, which twists the carbon chain toward the opposite side of the ribose from the adenine ring. This "bent" conformation oriented the activated methyl group so that it pointed toward the catalytic DPPY (65–68) and the active site of the enzyme (see Fig. 3). The C4′-C5′-SD-CG torsion angles were -169° and -143° for AdoHcy and sinefungin, respectively. These angles placed the tail portion of the ligand (homocysteine for AdoHcy or ornithine for sinefungin) in the same plane as the ribose (Fig. 2). This "extended" conformation permits direct interaction of the tail α-amino and α-carbonyl groups with the catalytic DPPY motif, an interaction that is not observed in the AdoMet complex. In the extended conformation, the ϵ-amino group of sinefungin, corresponding to the activated methyl group in AdoMet, was oriented toward the exterior of the protein and formed part of the solvent-exposed surface, unlike the methyl group of AdoMet (Fig. 3A). The binding pocket contains water molecules, and a chloride ion sits at the position occupied by a carboxylate oxygen atom of the methionine tail (Fig. 2). A second chloride forms a hydrogen bond to the backbone amide of Leu137 and to the side chains of Asn136 and Asn206 near the putative target recognition domain (TRD) of the protein (residues 165–210). Using anomalous scattering from bromide-soaked M.RsrI-AdoHcy co-crystals, we observed substitution of the second chloride by bromide, confirming that this site binds negative ions. The chloride ion in the ligand-binding pocket did not exchange with bromide, but it is likely that access to this site was restricted by the bound AdoHcy.Fig. 1Ligands of DNA methyltransferases. Shown are AdoMet; AdoHcy, sinefungin, and 5′-MTA. The tails attached to the C-5′ of the adenosine moiety (R) are shown individually, with atom labels shown for AdoMet.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (20Schluckebier G. Kozak M. Bleimling N. Weinhold E. Saenger W. J. Mol. Biol. 1997; 265: 56-67Crossref PubMed Scopus (111) Google Scholar).Table IIIInteractions of AdoMet, AdoHcy, and sinefungin with M.RsrILigand atomsAdoMet complexAdoHcy complexSinefungin complexN1Cys47 (N*), Asp46, Ala272Same + Cys45Same + Phe250C2Cys45, Cys47, Phe249, Ala272, Phe250Same + Asp46SameN3Ala272 (N*), Phe249, Phe250Same + Asp271SameC4Phe250, Ala272SameSameC5Phe250, Ala272Phe250Phe250C6Phe250, Ala272, Asp46Phe250, Asp46Phe250, Asp46N6Asp46 (OD2*), Trp84Asp46 (OD2*)SameN7Pro67NoneSameC8Pro67NoneSameN9Phe250SameSameC1′Asp271SameSameC2′Asp271SameSameO2′Asp271(OD1*), Ala273SameSameC3′His223, Asp271SameSameO3′His223 (NE2*), Asp271(OD2*), Gly252SameSameC4′Phe250, Asp271Asp271Asp271O4′Phe250, Asp271SameSameC5′Asp65Asp65Pro67CGAsp65, Phe250Asp65, Pro66, Thr225Asp65, Pro66SDAsp65, His223, Thr225(OG1*)Asp65, His223, Thr225CD-Thr225CEAsp65, Thr225NANE-Thr225(OG1*)CBAsp65, Thr225SameSameCAAsp65, Ala251Asp65Asp65, Pro66NAsp65(OD2*), Lys227Asp65(OD1*), Pro66(O*)Asp65(OD1*), Pro66(O*), Tyr68CThr225, Gln226, Lys227, Ala251, Ser253NoneNoneCO1Gln226, Ala251, Gly252, Ser253(N*, OG*)Pro66Pro66CO2Thr225, Gln226, Lys227(N*), Ser253, Val255Thr225Thr225 Open table in a new tab 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 (6Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (170) Google Scholar). 2X. Cheng, personal communication. 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.Table IVLigand torsion angles in methyltransferase structuresStructureTail torsion anglesaSee Fig. 1 for atom designationsSugar configurationsAdoMetO4′-C1′-N9-C4O4′-C4′-C5′-SDC4′-C5′-SD-CGC5′-SD-CG-CBSD-CG-CB-CACG-CB-CA-CCB-CA-C-O1M.RsrI-99176-70168177160-53C1′ exoM.TaqI-AbM.TaqI crystallized with two proteins in the asymmetric unit and each protein was analyzed independently-79158-82103169-7288C1′ exoM.TaqI-BbM.TaqI crystallized with two proteins in the asymmetric unit and each protein was analyzed independently-83154-68109168-86-62C2′ exoM.DpnII2-97178-66-171179162-87C1′ exoM.HhaI-123171-37-13617083139C1′ exo/C2′ endoSinefunginO4′-C1′-N9-C4O4′-C4′-C5′-CDC4′-C5′-CD-CGC5′-CD-CG-CBCD-CG-CB-CACG-CB-CA-CCB-CA-C-O1M.RsrI-100173-143-178154-8286C2′ endoM.TaqI-AbM.TaqI crystallized with two proteins in the asymmetric unit and each protein was analyzed independently-1023417355169176-94C3′ endoM.TaqI-BbM.TaqI crystallized with two proteins in the asymmetric unit and each protein was analyzed independently-10016-177157-16177-94C4′ exo/C3′ endoAdoHcyO4′-C1′-N9-C4O4′-C4′-C5′-SDC4′-C5′-SD-CGC5′-SD-CG-CBSD-CG-CB-CACG-CB-CA-CCB-CA-C-O1M.RsrI-95172-169157-178-66114C2′ endo/C1′ exoM.TaqI-AbM.TaqI crystallized with two proteins in the asymmetric unit and each protein was analyzed independently-10583175-127-179-61-85C3′ endoM.TaqI-BbM.TaqI crystallized with two proteins in the asymmetric unit and each protein was analyzed independently-10672162146-17776-56C2′ endoM.PvuII-10315953127170140-93O4′ endoa See Fig. 1 for atom designationsb M.TaqI crystallized with two proteins in the asymmetric unit and each protein was analyzed independently Open table in a new tab 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 α-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 (14Scavetta R.D. Thomas C.B. Walsh M.A. Szegedi S. Joachimiak A. Gumport R.I. Churchill M.E.A. Nucleic Acids Res. 2000; 28: 3950-3961Crossref PubMed Google Scholar). The differences between the AdoMet structure and
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