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Recognition of Flanking DNA Sequences by EcoRV Endonuclease Involves Alternative Patterns of Water-mediated Contacts

1998; Elsevier BV; Volume: 273; Issue: 34 Linguagem: Inglês

10.1074/jbc.273.34.21721

ISSN

1083-351X

Autores

Nancy C. Horton, John J. Perona,

Tópico(s)

RNA Research and Splicing

Resumo

The 2.1-Å cocrystal structure ofEcoRV endonuclease bound to 5′-CGGGATATCCC, in a crystal lattice isomorphous with the cocrystallized undecamer 5′-AAAGATATCTT previously determined, shows novel base recognition in the major groove of the DNA flanking the GATATC target site. Lys104 of the enzyme interacts through water molecules with the exocyclic N-4 amino groups of flanking cytosines. Steric exclusion of water molecule-binding sites by the 5-methyl group of thymine drives the adoption of alternative water-mediated contacts with ATversus GC flanks. This structure provides a rare example of structural adaptability in the recognition of different DNA sequences by a protein and suggests preferred strategies for the expansion of target site specificity by EcoRV. The 2.1-Å cocrystal structure ofEcoRV endonuclease bound to 5′-CGGGATATCCC, in a crystal lattice isomorphous with the cocrystallized undecamer 5′-AAAGATATCTT previously determined, shows novel base recognition in the major groove of the DNA flanking the GATATC target site. Lys104 of the enzyme interacts through water molecules with the exocyclic N-4 amino groups of flanking cytosines. Steric exclusion of water molecule-binding sites by the 5-methyl group of thymine drives the adoption of alternative water-mediated contacts with ATversus GC flanks. This structure provides a rare example of structural adaptability in the recognition of different DNA sequences by a protein and suggests preferred strategies for the expansion of target site specificity by EcoRV. Restriction endonucleases function in all prokaryotes as components of defensive restriction-modification systems and are superb models for the study of protein-DNA interactions owing to their exceptionally high sequence specificities. The type II restriction-modification systems are the best studied from a structure-function perspective and are composed of a homodimeric endonuclease and monomeric methylase (1Roberts R.J. Halford S.E. Linn S.M. Lloyd R.S. Roberts R.J. Nucleases. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 35-88Google Scholar, 2Pingoud A. Jeltsch A. Eur. J. Biochem. 1997; 246: 1-22Crossref PubMed Scopus (302) Google Scholar). The crystal structures of the following six restriction enzymes have been determined:EcoRI (3Rosenberg J. Curr. Opin. Struct. Biol. 1991; 1: 104-113Crossref Scopus (156) Google Scholar), EcoRV (4Winkler F.K. Banner D.W. Oefner C. Tsernoglou D. Brown R.S. Heathman S.P. Bryan R. Martin P.D. Petratos K. Wilson K.S. EMBO J. 1993; 12: 1781-1795Crossref PubMed Scopus (445) Google Scholar, 5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar, 6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar), BamHI (7Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Structure. 1994; 2: 439-452Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 8Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Science. 1995; 269: 656-663Crossref PubMed Scopus (291) Google Scholar),PvuII (9Cheng X. Balendiran K. Schildkraut I. Anderson J.E. EMBO J. 1994; 13: 3927-3935Crossref PubMed Scopus (198) Google Scholar), Cfr10I (10Bozic D. Grazulis S. Siksnys V. Huber R. J. Mol. Biol. 1996; 255: 176-186Crossref PubMed Scopus (92) Google Scholar), and FokI (11Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A. Nature. 1997; 388: 97-100Crossref PubMed Scopus (214) Google Scholar). With the exception of Cfr10I and EcoRI, the enzymes have been solved in both the absence and presence of DNA. The structures reveal extensive complementarity at the protein-DNA interfaces, which appears to explain the high specificities of up to 106-fold in cleavage rate constants for the specific sites (Ref. 12Jen-Jacobson L. Methods Enzymol. 1995; 259: 305-344Crossref PubMed Scopus (41) Google Scholar and references therein). An additional, less appreciated, contribution to specificity may also arise from DNA-induced conformational changes in the enzymes. EcoRV has recently emerged as the best studied of the restriction endonucleases from both structural and mechanistic standpoints. This enzyme cleaves the sequence 5′-GATATC-3′ at the center TA step in a blunt-ended fashion, generating 5′-phosphate groups (13Schildkraut I. Banner C.D.B. Rhodes C.S. Parekh S. Gene ( Amst .). 1984; 27: 327-329Crossref PubMed Scopus (59) Google Scholar). The cocrystal structure of the EcoRV-DNA complex reveals a tight network of hydrogen bonding and electrostatic and van der Waals contacts at the protein-DNA interface over the entire hexameric DNA site (4Winkler F.K. Banner D.W. Oefner C. Tsernoglou D. Brown R.S. Heathman S.P. Bryan R. Martin P.D. Petratos K. Wilson K.S. EMBO J. 1993; 12: 1781-1795Crossref PubMed Scopus (445) Google Scholar). Specificity at the outer two base pairs of each half-site is determined by hydrogen bonding with discriminating base functional groups in the major groove. At the center step the DNA is bent sharply by 50° into the major groove, so that protein cannot penetrate to contact the hydrogen bonding moieties. Indirect readout is thus implicated in specificity at this position. This may originate in part from differences in the energetic cost of partially unstacking the center TA step relative to CG or GC steps (14Horton N.C. Perona J.J. J. Mol. Biol. 1998; 277: 779-787Crossref PubMed Scopus (40) Google Scholar). Whereas the specificity of EcoRV in vivo is limited to the hexamer target site GATATC, the crystal structures show that the enzyme also contacts 2–3 base pairs of DNA to either side of this site (4Winkler F.K. Banner D.W. Oefner C. Tsernoglou D. Brown R.S. Heathman S.P. Bryan R. Martin P.D. Petratos K. Wilson K.S. EMBO J. 1993; 12: 1781-1795Crossref PubMed Scopus (445) Google Scholar). These contacts with flanking DNA in five cocrystal structures with 5′-AAAGATATCTT at 2.0–2.1-Å resolution (5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar, 6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar, 14Horton N.C. Perona J.J. J. Mol. Biol. 1998; 277: 779-787Crossref PubMed Scopus (40) Google Scholar), and with the decamer 5′-GGGATATCCC at 3.0 Å, are primarily with the sugar-phosphate backbone (4Winkler F.K. Banner D.W. Oefner C. Tsernoglou D. Brown R.S. Heathman S.P. Bryan R. Martin P.D. Petratos K. Wilson K.S. EMBO J. 1993; 12: 1781-1795Crossref PubMed Scopus (445) Google Scholar, 5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar). Several studies have shown that the enzyme is sensitive to perturbation of these contacts. Replacement of phosphate groups with phosphorothioates showed thatSp and Rp substitutions directly 3′ to GATATC reduceVmax/Km toward a dodecamer substrate by 4- and 50-fold, respectively (15Thorogood H. Grasby J.A. Connolly B.A. J. Biol. Chem. 1996; 271: 8855-8862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Furthermore, mutation of four amino acids contacting flanking phosphate groups reduceskcat/Km toward a 20-mer substrate containing GATATC (16Wenz C. Jeltsch A. Pingoud A. J. Biol. Chem. 1996; 271: 5565-5573Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The most striking effect occurs with the mutant R226A within the C-terminal subdomain, which lowers activity by nearly 103-fold relative to the wild-type enzyme. These studies show that the distal enzyme-DNA contacts outside the target site significantly increase the catalytic rate, perhaps by helping to orchestrate the mutual conformational changes in the enzyme and DNA which occur en route to the transition state. The biological role of EcoRV (and other type II enzymes) requires that all flanking sequences permit target site cleavage with high catalytic efficiency. However, this does not preclude the existence of some sequence preference. Indeed, 10- and 500-fold variations in binding constants with different flanks have been found in in vitrostudies of EcoRV and EcoRI, respectively (17Engler L.E. Welch K.K. Jen-Jacobson L. J. Mol. Biol. 1997; 269: 82-101Crossref PubMed Scopus (96) Google Scholar,18Jen-Jacobson L. Biopolymers. 1997; 44: 153-180Crossref PubMed Scopus (172) Google Scholar). Additionally, within the context of cleavage of the "star" site GTTATC, EcoRV prefers 5′-G and 3′-C on either side of the target (19Taylor J.D. Halford S.E. Biochemistry. 1989; 28: 6198-6207Crossref PubMed Scopus (119) Google Scholar, 20Taylor J.D. Halford S.E. Biochemistry. 1992; 31: 90-97Crossref PubMed Scopus (46) Google Scholar). These flanking sequence selectivities are modest compared with the 106-fold and greater specificitiesversus base substitutions internal to the GATATC site, but they nonetheless suggest that target site expansion may be feasible. This is a prospect of considerable practical importance for the development of new restriction enzymes specific for 8–10 base recognition sites. Attempts to alter the substrate specificity of the EcoRI andEcoRV restriction endonucleases have not been successful thus far (21Heitman J. Model P. EMBO J. 1990; 9: 3369-3378Crossref PubMed Scopus (53) Google Scholar, 22Flores H. Osuna J. Heitman J. Soberon X. Gene ( Amst .). 1995; 157: 295-301Crossref PubMed Scopus (25) Google Scholar, 23Wenz C. Selent U. Wende W. Jeltsch A. Wolfes H. Pingoud A. Biochim. Biophys. Acta. 1994; 1219: 73-80Crossref PubMed Scopus (37) Google Scholar, 24Lanio T. Selent U. Wenz C. Wende W. Schulz A. Adiraj M. Katti S.B. Pingoud A. Protein Eng. 1996; 9: 1005-1010Crossref PubMed Google Scholar), indicating a need for further basic studies. WhereasEcoRV is the best studied enzyme of the class, its structure bound to DNA with GC flanks is determined at only 3.0 Å resolution (4Winkler F.K. Banner D.W. Oefner C. Tsernoglou D. Brown R.S. Heathman S.P. Bryan R. Martin P.D. Petratos K. Wilson K.S. EMBO J. 1993; 12: 1781-1795Crossref PubMed Scopus (445) Google Scholar). This is insufficient for describing detailed protein-DNA interactions, particularly those involving solvent molecules. Moreover, large differences in DNA and protein conformation, most probably arising from altered crystal packing contacts, have been noted in the structures of the enzyme bound to specific DNA sites possessing ATversus GC flanks (4Winkler F.K. Banner D.W. Oefner C. Tsernoglou D. Brown R.S. Heathman S.P. Bryan R. Martin P.D. Petratos K. Wilson K.S. EMBO J. 1993; 12: 1781-1795Crossref PubMed Scopus (445) Google Scholar, 5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar, 6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar, 14Horton N.C. Perona J.J. J. Mol. Biol. 1998; 277: 779-787Crossref PubMed Scopus (40) Google Scholar). Therefore, to characterize the structural aspects of the flanking interactions better, we have determined a new cocrystal structure of EcoRV bound to 5′-CGGGATATCCC at 2.1 Å resolution. We find that Lys104makes novel water-mediated interactions with hydrogen bonding functional groups of the flanking GC bases. By contrast, no interactions of Lys104 with bases of the flanking DNA are present in the five 2.0–2.1-Å resolution complexes with 5′-AAAGATATCTT (5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar, 6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar, 14Horton N.C. Perona J.J. J. Mol. Biol. 1998; 277: 779-787Crossref PubMed Scopus (40) Google Scholar). These data provide insight into new modes of flanking sequence recognition by EcoRV and suggest preferred alternatives for engineering sequence specificity for larger DNA sites into the enzyme. Wild-type EcoRV was prepared as described and stored as an ammonium sulfate pellet at 4 °C (6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar). The self-complementary oligonucleotide 5′-CGGGATATCCC was synthesized for cocrystallization by standard methods and purified on a Rainin PureDNA high pressure liquid chromatography column developed in a gradient of triethylammonium acetate/acetonitrile. Detritylation was performed on the column (25Aggarwal A.K. Methods: A Companion to Methods in Enzymology. 1990; 1: 83-90Crossref Scopus (63) Google Scholar). The DNA was lyophilized and stored at −20 °C until ready for use, when it was brought to a concentration of 10 mg/ml in 50 mm Tris (pH 7.5), 1 mm EDTA. Cocrystals ofEcoRV complexed with 5′-CGGGATATCCC were grown by vapor diffusion from solutions containing 0.17 mm EcoRV and 0.34 mm DNA, in the presence of 15% PEG 4K, 100 mm imidazole (pH 6.5), and 150 mmNaCl (final conditions). The protein was prepared by resuspending the ammonium sulfate slurry at 30 mg/ml in a buffer containing 10 mm HEPES (pH 7.5), 250 mm NaCl, 1 mm EDTA, and 0.1 mm dithiothreitol and then exhaustively dialyzed versus this buffer. Crystals were mounted for data collection directly from the drop. X-ray diffraction amplitudes were measured on an R-AXIS IIC area detector mounted on a Rigaku RU-200 rotating anode generator. Data were obtained at ambient temperature from two crystals. Determination of the orientation matrix, integration, scaling, and merging of data was performed with DENZO and with the HKL suite of programs (26Otwinowski Z. Proceedings of the CCP 4 Study Weekend: Data Collection and Processing, January, 29–30, 1993.in: Sawyer L. Isaacs N. Bailey S. SERC Daresbury Laboratory, England1993: 56-62Google Scholar). Local scaling of the structure-factor amplitudes against those of the isomorphous cocrystal with 5′-AAAGATATCTT (6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar) was performed using the program MAXSCALE. The resulting scaled data set resulted in a refined structure possessing anRfree (Table I) reduced by 2% as compared with the refinement versus unscaled data.Table ICrystallographic data collection and refinement statisticsDNA sequenceResolutionSpace groupCell dimensionsData coverageaIncludes all data in the intensity range I/ς(I) > −3.0.RmergeBoverbOverall B factor is determined from a Wilson plot of the structure factor data using a low resolution cut-off of 4.7 Å.RcrystcRefinement was carried out using a low resolution cut-off of 6.0 Å.Rfreer.m.s. bondsr.m.s. angNo. watersabcαβγÅÅ%Å2ÅCGGGATATCCC2.1P149.450.264.196°109°108°850.05327.40.1960.2940.0121.86°236Rmerge = (∑h∑i‖〈Fh〉 −Fhi‖)/(∑h Fh) where 〈Fh〉 is the mean structure factor magnitude ofi observations of symmetry-related reflections with Bragg index h. Rcryst = (∑h∑i‖ ‖Fobs‖ − ‖Fcalc‖ ‖)/(∑‖Fobs‖) where Fobs and Fcalc are the observed and calculated structure factor magnitudes. Rfree is calculated with removal of 10% of the data as the test set, followed by simulated annealing refinement of the final model. r.m.s. indicates root mean square.a Includes all data in the intensity range I/ς(I) > −3.0.b Overall B factor is determined from a Wilson plot of the structure factor data using a low resolution cut-off of 4.7 Å.c Refinement was carried out using a low resolution cut-off of 6.0 Å. Open table in a new tab Rmerge = (∑h∑i‖〈Fh〉 −Fhi‖)/(∑h Fh) where 〈Fh〉 is the mean structure factor magnitude ofi observations of symmetry-related reflections with Bragg index h. Rcryst = (∑h∑i‖ ‖Fobs‖ − ‖Fcalc‖ ‖)/(∑‖Fobs‖) where Fobs and Fcalc are the observed and calculated structure factor magnitudes. Rfree is calculated with removal of 10% of the data as the test set, followed by simulated annealing refinement of the final model. r.m.s. indicates root mean square. The crystal structure of EcoRV complexed to 5′-CGGGATATCCC was phased directly using the isomorphous cocrystal with 5′-AAAGATATCTT (5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar) as the starting model. The flanking DNA sequences and 57 water molecules at the enzyme-DNA interface were removed from the model. The starting crystallographic R-factor was 31.1%, and this was reduced to 19.6% by several rounds of model building iterated with positional B-factor and simulated annealing refinement in XPLOR (27Brunger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar). Very tight stereochemical constraints were maintained throughout the refinement. Criteria for the inclusion of water molecules in both structures were the appearance of peaks at 1.0 ς in (2Fo − Fc) maps, 3.0 ς in (Fo − Fc) maps, and at least one hydrogen bonding interaction with protein or DNA. All water molecules and side chains possessing B-factors above 50.0 Å2 were carefully examined prior to inclusion in the final model. Model building utilized the programs CHAIN (28Sack J.S. J. Mol. Graphics. 1988; 6: 224-225Crossref Google Scholar) and LORE (29Finzel B.C. Acta Crystallogr. D. 1995; 51: 450-457Crossref PubMed Google Scholar). Parameters for the DNA used in XPLOR refinement were those recently described (30Parkinson G. Vojtechovsky J. Clowney L. Brunger A.T. Berman H.M. Acta Crystallogr. D. 1996; 52: 57-64Crossref PubMed Scopus (268) Google Scholar). Determination of Cα atoms in the dimerization or DNA-binding domains was by difference-distance calculations, as described (6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar). Least squares superpositions were performed with Insight II (31Dayringer H. Tramontano A. Sprang S. Fletterick R.J. J. Mol. Graphics. 1986; 4: 82Crossref Scopus (164) Google Scholar) and GEM (32Perry K.M. Fauman E.B. Finer-Moore J.S. Montfort W.R. Maley G.F. Maley F. Stroud R.M. Proteins. 1990; 8: 315-333Crossref PubMed Scopus (152) Google Scholar). EcoRV was cocrystallized with the undecamer duplex 5′-CGGGATATCCC possessing a 5′-C overhang (dGC) 1The abbreviations used are: dGCcocrystal structure of EcoRV bound to 5′-CGGGATATCCCdATcocrystal structure of EcoRV bound to 5′-AAAGATATCTTWatwater. in the absence of divalent metals. The crystals are in the well described P1 lattice (5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar,6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar) with cell dimensions isomorphous to the previous complexes (TableI). Nearly all of the molecular packing interactions in this crystal are identical with those of the cocrystal with 5′-AAAGATATCTT (dAT). Only base stacking of the unpaired 5′ nucleotide (C versus A) on the dimerization domain of an adjoining molecule is very slightly altered because of the differing sizes of the pyrimidine and purine rings. The crystals have an enzyme dimer and duplex DNA in the asymmetric unit, so that this packing interaction is not made on the opposite end of the molecule. Conserved features observed on each of the two flanks occur in the context of different nearby lattice contacts and are consequently likely to reflect true aspects of the flanking interactions in solution. Thus, comparison of the cocrystal structures of EcoRV complexed with dAT versus dGC provides an excellent opportunity to elucidate detailed differences in the interactions of EcoRV with AT versus GC flanks. cocrystal structure of EcoRV bound to 5′-CGGGATATCCC cocrystal structure of EcoRV bound to 5′-AAAGATATCTT water. The overall structure of this EcoRV-DNA complex is very similar to that of EcoRV cocrystallized with dAT (Fig.1 (5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar)), with no significant differences in structure detectable within the GATATC target site. The quaternary structure is also identical with that of the dAT cocrystal lacking divalent metal ions. The relative orientations of the DNA binding/catalytic domains in the two structures differ by only a 0.4–0.6° rotation and a 0.22-Å difference in the center of mass separation of the two subunits. These differences are within the level of coordinate error, estimated from Luzzatti plots at roughly 0.2–0.25 Å for each structure (data not shown). Small but perhaps significant intersubunit rotations of 1.0–1.5° are present in comparisons of either cocrystal lacking divalent metals, with isomorphous crystals of metal-containing ternary complexes (data not shown (5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar, 6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar)). Nearly all of the previously observed major and minor groove enzyme-DNA contacts, both with base functional groups and sugar-phosphate moieties, are also present in this complex. The only significant exception is the conformation of the Arg221 side chain in both subunits. In the dAT structure the guanidinium group makes a direct electrostatic interaction with a DNA phosphate at GpATATC in subunit I and a water-mediated interaction with the identical phosphate on the opposite half-site in subunit II (5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar). In the dGC structure, the direct contact is absent, and the side chain instead adopts a novel well ordered conformation where it bridges through two ordered water molecules to the guanidinium group of Arg115 (Fig.2). The water-mediated phosphate interaction in subunit II is preserved in dGC, but the conformation of Arg221 differs somewhat to permit an additional water-mediated interaction with Arg115. The conformations observed in dGC are further demonstrations of the alternative binding modes accessible to Arg221 and are unique in the sense that in one subunit no contacts with the DNA are made. Apparently Arg221 has very little energetic preference for making water-mediated intramolecular protein contacts as compared with interactions with the DNA. Conformations of the surrounding 220s loop, the DNA backbone, and Arg115 are identical in the two structures. The mutation R221A is without significant effect on the activity of the enzyme (16Wenz C. Jeltsch A. Pingoud A. J. Biol. Chem. 1996; 271: 5565-5573Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and this can now be rationalized based on the observation that the side chain adopts conformations which do not involve DNA binding. The newly introduced exocyclic amino groups of the guanosine flanking bases are outside of the region contacted by the minor groove bindingQ loops (Fig. 1) and surface loops at residues Lys119-Asn120 and are consequently without effect on the structure. However, differences between the dAT and dGC cocrystal structures appear in the major groove interactions of the flanking base pairs with the enzyme. On both sides of the GATATC target the exocyclic N-4 amino groups of flanking cytosines interact through water molecules to Lys104 on an enzyme surface loop (Fig.3, A and B). Water molecules bridging Lys104 to the flanking bases are not present in any of the dAT structures previously determined (5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar, 6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar) and could not be visualized in the cocrystal with the decamer 5′-GGGATATCCC (4Winkler F.K. Banner D.W. Oefner C. Tsernoglou D. Brown R.S. Heathman S.P. Bryan R. Martin P.D. Petratos K. Wilson K.S. EMBO J. 1993; 12: 1781-1795Crossref PubMed Scopus (445) Google Scholar) owing to the lower resolution (3 Å) of this structure. In subunit I Lys104 bridges through a network of water molecules binding in the major groove at the two flanking CG pairs (Fig. 3 A and Table II). The water molecules hydrogen-bond with protein at three positions as follows: the Lys104 side chain amine, the backbone at amino acids Ala181-Gly182, and the backbone at Phe105. Water molecules 3 and 5 also bridge through other water molecules to the DNA phosphates at Cyt1 and Cyt9 (Table II). In this subunit Lys104interacts through one water molecule to the N-4 of Cyt11and through two water molecules to the O-6 of its base-pairing partner at Gua2, whereas its connection to the inner flanking pair Gua3-Cyt10 is through a chain of three water molecules: Wat1,3,4. Recognition of this inner pair occurs via water molecules more closely connected to the Ala181-Gly182 main chain. The Gly182-amide also serves as a hydrogen bond acceptor from the N-4 of Cyt9, providing part of the discrimination for the outer GC base pairs of GATATC (Fig. 3,A and B). Similar water-mediated interactions of Ala181 and Gly182 with the inner Ade3-Thy10 pair are observed in the dAT structures (4Winkler F.K. Banner D.W. Oefner C. Tsernoglou D. Brown R.S. Heathman S.P. Bryan R. Martin P.D. Petratos K. Wilson K.S. EMBO J. 1993; 12: 1781-1795Crossref PubMed Scopus (445) Google Scholar, 5Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar, 6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar, 14Horton N.C. Perona J.J. J. Mol. Biol. 1998; 277: 779-787Crossref PubMed Scopus (40) Google Scholar). However, in these structures no direct or water-mediated recognition of base functional groups on the outer flanking Ade2-Thy11 pair occurs.Table IIInteractions of water molecules mediating flanking sequence recognitionSubunit ISubunit IIAtomDistanceAtomDistanceÅÅWat1Wat1′ Lys104:Nε2.8 Lys104:N ε2.5 Cyt11:N43.2 Wat2′3.0 Wat22.6 Wat32.6Wat2Wat2′ Wat12.6 Cyt10:N-42.7 Cyt11:N-42.9 Wat1′3.0 Wat3′3.1Wat3Wat3′ Gua2:O-62.7 Gly182:O3.2 Wat12.6 Wat2′3.1 Wat42.6 WatbThis water bridges through other water molecules to the main chain amides of Lys104 and Phe105 and to the DNA phosphate at C-9.2.8 WataThis water bridges through one other water molecule to the side chain of His193.2.7Wat4 Cyt10:N-43.2 Wat32.6 Wat52.6Wat5 Gly182:O3.0 Wat42.6 Wat62.5 WatcThis water bridges through one other water molecule to the DNA phosphate at C-9.2.7Wat6 Phe105:O2.7 Wat52.5 Wat73.2Wat7 Ala181:O2.6 Wat63.2a This water bridges through one other water molecule to the side chain of His193.b This water bridges through other water molecules to the main chain amides of Lys104 and Phe105 and to the DNA phosphate at C-9.c This water bridges through one other water molecule to the DNA phosphate at C-9. Open table in a new tab In subunit II the Lys104-amine group interacts directly with two water molecules, one of which (Wat2′) bridges directly to the N-4 amino group of the inner flanking cytosine (Fig.3 B). Wat2′ also interacts with another water (Wat3′) which donates a hydrogen bond to the main chain amide of Gly182. While preserving the common feature of water-mediated flanking sequence recognition through Lys104, the interactions made in subunits I and II thus clearly differ. This appears attributable to the proximity of a crystal packing contact made by the 5-overhanging cytosine (Cyt1) in subunit I. The base of Cyt1 packs onto the peptide main chain in the dimerization domain of an adjoining molecule, which provides stabilization in a manner apparently similar to the continuation of base-stacking in a longer duplex DNA. In subunit II, which lacks this lattice contact, the 5′-overhanging cytosine is disordered. Moreover, the outer flanking Gua2-Cyt11 pair is also destabilized; atomicB-factors of Cyt11 are above 60 Å2, and the electron density for this nucleotide is weak in the final 2Fo − Fc electron density maps (Fig. 3 D). Superposition of the flanking interactions from subunits I and II of dGC shows that Cyt11 of subunit II is shifted away from the protein by approximately 1.0–1.5 Å (Fig.4), and this can account for why water-mediated interactions with the Gua2-Cyt11pair are not observed in subunit II. It thus appears that the water-mediated flanking interactions in subunit I are more likely to be representative of the contacts present in solution. The recognition of the flanking CG base pairs by these water-mediated interactions is clearly nonspecific. In no case does a water molecule make more than one hydrogen bond with protein, a requirement for presenting obligate donor/acceptor functions to the DNA (33Steitz T.A. Q. Rev. Biophys. 1990; 23: 205-280Crossref PubMed Scopus (462) Google Scholar). Therefore, the waters could in principle reorient their two hydrogen bond donor and acceptor groups to provide equivalent interactions with flanking TA pairs. Moreover, it is also very unlikely that discrimination could be achieved against flanking GC or AT base pairs in which the purine and pyrimidine rings are exchanged. This is because the positions of the major groove hydrogen-bonding sites in these pairs are altered by only 1 Å (34Seeman N.C. Rosenberg J.M. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 804-808Crossref PubMed Scopus (951) Google Scholar), and this shift should be readily accommodated by small rearrangements of the waters. To address why the apparently nonspecific water-mediated interactions between Lys104 and the outer flanking base pairs are not present in dAT, we superimposed the dAT and dGC structures based on polypeptide backbone atoms within the DNA-binding domains (root mean square deviation = 0.24 Å for the superposition of 244 amino acids (6Perona J.J. Martin A.M. J. Mol. Biol. 1997; 273: 207-225Crossref PubMed Scopus (68) Google Scholar)). This shows that in both subunits one of the water molecules bound to Lys104 is blocked from binding to flanking AT pairs by one of the thymine C-5 methyl groups (Fig.5, A and B). In subunit II the steric hindrance by Thy10, occluding binding of Wat2′, is sufficient to acco

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