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• W2133187258 abstract "Nonspecific, extracellular nucleases have received enhanced attention recently as a consequence of the critical role that these enzymes can play in infectivity by overcoming the host neutrophil defense system. The activity of the cyanobacterial nuclease NucA, a member of the ββα Me superfamily, is controlled by the specific nuclease inhibitor, NuiA. Here we report the 2.3-Aå resolution crystal structure of the NucA-NuiA complex, showing that NucA inhibition by NuiA involves an unusual divalent metal ion bridge that connects the nuclease with its inhibitor. The C-terminal Thr-135NuiA hydroxyl oxygen is directly coordinated with the catalytic Mg2+ of the nuclease active site, and Glu-24NuiA also extends into the active site, mimicking the charge of a scissile phosphate. NuiA residues Asp-75 and Trp-76 form a second interaction site, contributing to the strength and specificity of the interaction. The crystallographically defined interface is shown to be consistent with results of studies using site-directed NuiA mutants. This mode of inhibition differs dramatically from the exosite mechanism of inhibition seen with the DNase colicins E7/E9 and from other nuclease-inhibitor complexes that have been studied. The structure of this complex provides valuable insights for the development of inhibitors for related nonspecific nucleases that share the DRGH active site motif such as the Streptococcus pneumoniae nuclease EndA, which mediates infectivity of this pathogen, and mitochondrial EndoG, which is involved in recombination and apoptosis. Nonspecific, extracellular nucleases have received enhanced attention recently as a consequence of the critical role that these enzymes can play in infectivity by overcoming the host neutrophil defense system. The activity of the cyanobacterial nuclease NucA, a member of the ββα Me superfamily, is controlled by the specific nuclease inhibitor, NuiA. Here we report the 2.3-Aå resolution crystal structure of the NucA-NuiA complex, showing that NucA inhibition by NuiA involves an unusual divalent metal ion bridge that connects the nuclease with its inhibitor. The C-terminal Thr-135NuiA hydroxyl oxygen is directly coordinated with the catalytic Mg2+ of the nuclease active site, and Glu-24NuiA also extends into the active site, mimicking the charge of a scissile phosphate. NuiA residues Asp-75 and Trp-76 form a second interaction site, contributing to the strength and specificity of the interaction. The crystallographically defined interface is shown to be consistent with results of studies using site-directed NuiA mutants. This mode of inhibition differs dramatically from the exosite mechanism of inhibition seen with the DNase colicins E7/E9 and from other nuclease-inhibitor complexes that have been studied. The structure of this complex provides valuable insights for the development of inhibitors for related nonspecific nucleases that share the DRGH active site motif such as the Streptococcus pneumoniae nuclease EndA, which mediates infectivity of this pathogen, and mitochondrial EndoG, which is involved in recombination and apoptosis. Nonspecific nucleases are involved in a broad range of functions that include extra- and intracellular digestion, programmed cell death, defense, replication, recombination, and repair (1Linn S. Lloyd R.S. Roberts R.J. Nucleases, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1993; Google Scholar, 2D'Alessio G. Riordan J.F. Ribonucleases: Structures and Functions. 1997; (Academic Press, San Diego)Google Scholar, 3Rangarajan E.S. Shankar V. FEMS Microbiol. Rev. 2001; 25: 583-613Crossref PubMed Google Scholar). They also have proven useful for determining nucleic acid structures, mapping mutations, studying the interaction of DNA and RNA with various ligands (4Schein C. Nuclease Methods and Protocols. 2001; (Humana Press Inc.)Crossref Google Scholar), and sequencing of RNA (5Krupp G. Gross H.J. Nucleic Acids Res. 1979; 6: 3481-3490Crossref PubMed Scopus (62) Google Scholar). Most recently, an important role for these nucleases in microbial infectivity has been demonstrated, based on their ability to digest the DNA component of host neutrophil extracellular traps (6Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6115) Google Scholar, 7Beiter K. Wartha F. Albiger B. Normark S. Zychlinsky A. Henriques-Normark B. Curr. Biol. 2006; 16: 401-407Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 8Buchanan J.T. Simpson A.J. Aziz R.K. Liu G.Y. Kristian S.A. Kotb M. Feramisco J. Nizet V. Curr. Biol. 2006; 16: 396-400Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar). Consequently, these nucleases are now recognized as significant drug targets, and information related to the inhibition of these enzymes is of potential use for inhibitor development. As a result of their ability to degrade nucleic acids nonspecifically, they also represent an endogenous toxic challenge. Therefore, regulation of their activity is critical for the cells that produce them. The ββα Me superfamily of nucleases (9Kuhlmann U.C. Moore G.R. James R. Kleanthous C. Hemmings A.M. FEBS Lett. 1999; 463: 1-2Crossref PubMed Scopus (99) Google Scholar) comprises nonspecific, structure-specific, and sequence-specific enzymes that share a structurally conserved active site scaffold and utilize a divalent metal ion. They can be grouped according to sequence motifs into three families: His-Cys box nucleases (e.g. I-PpoI (10Flick K.E. Jurica M.S. Monnat Jr., R.J. Stoddard B.L. Nature. 1998; 394: 96-101Crossref PubMed Scopus (191) Google Scholar)), HNH nucleases (e.g. colicins E7 and E9 (11Hsia K.C. Chak K.F. Liang P.H. Cheng Y.S. Ku W.Y. Yuan H.S. Structure (Camb.). 2004; 12: 205-214Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 12Mate M.J. Kleanthous C. J. Biol. Chem. 2004; 279: 34763-34769Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and I-HmuI (13Shen B.W. Landthaler M. Shub D.A. Stoddard B.L. J. Mol. Biol. 2004; 342: 43-56Crossref PubMed Scopus (81) Google Scholar)), and DRGH nucleases (e.g. the extracellular nuclease from Serratia marcescens (14Nestle M. Roberts W.K. J. Biol. Chem. 1969; 244: 5213-5218Abstract Full Text PDF PubMed Google Scholar), the DNA entry nuclease EndA from Streptococcus pneumoniae (15Puyet A. Greenberg B. Lacks S.A. J. Mol. Biol. 1990; 213: 727-738Crossref PubMed Scopus (75) Google Scholar), the Syncephalastrum racemosum nuclease (16Ho H.C. Liao T.H. Biochem. J. 1999; 339: 261-267Crossref PubMed Google Scholar), nuclease C1 from Cunninghamella echinulata (17Ho H.C. Shiau P.F. Liu F.C. Chung J.G. Chen L.Y. Eur. J. Biochem. 1998; 256: 112-118Crossref PubMed Scopus (18) Google Scholar), yeast Nuc1 (18Vincent R.D. Hofmann T.J. Zassenhaus H.P. Nucleic Acids Res. 1988; 16: 3297-3312Crossref PubMed Scopus (81) Google Scholar), mitochondrial EndoG (19Cote J. Renaud J. Ruiz-Carrillo A. J. Biol. Chem. 1989; 264: 3301-3310Abstract Full Text PDF PubMed Google Scholar), and the Anabaena nuclease NucA (20Muro-Pastor A.M. Flores E. Herrero A. Wolk C.P. Mol. Microbiol. 1992; 6: 3021-3030Crossref PubMed Scopus (65) Google Scholar)). Whereas the eukaryotic nucleases of the DRGH family represent the major mitochondrial nuclease activity, the prokaryotic members of this family are responsible for extracellular DNA degradation. Notably the DNA-entry nucleases EndA from Streptococcus pneumoniae and the related Streptodornase (Sda1) from Streptococcus sp. allow their host organisms to escape from neutrophil extracellular traps by digesting the DNA scaffold of these structures, thereby evading the first line of defense against microbial infection in mammals (6Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6115) Google Scholar, 7Beiter K. Wartha F. Albiger B. Normark S. Zychlinsky A. Henriques-Normark B. Curr. Biol. 2006; 16: 401-407Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 8Buchanan J.T. Simpson A.J. Aziz R.K. Liu G.Y. Kristian S.A. Kotb M. Feramisco J. Nizet V. Curr. Biol. 2006; 16: 396-400Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar). NucA, a member of the DRGH family, is one of the most potent nucleases known, and it degrades both single- and double-stranded DNA and RNA. Its activity is regulated by a potent and specific protein inhibitor, NuiA, which forms a tight 1:1 complex with picomolar affinity (21Meiss G. Gimadutdinow O. Haberland B. Pingoud A. J. Mol. Biol. 2000; 297: 521-534Crossref PubMed Scopus (33) Google Scholar). The structure of the active site is closely analogous to that of the Serratia nuclease (22Ghosh M. Meiss G. Pingoud A. London R.E. Pedersen L.C. J. Biol. Chem. 2005; 280: 27990-27997Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 23Miller M.D. Cai J. Krause K.L. J. Mol. Biol. 1999; 288: 975-987Crossref PubMed Scopus (86) Google Scholar), whereas the activity of the Serratia enzyme is dependent on the presence of cystine bonds and hence is determined by the redox level of the medium (24Ball T.K. Suh Y. Benedik M.J. Nucleic Acids Res. 1992; 20: 4971-4974Crossref PubMed Scopus (32) Google Scholar). A deletion analysis of NuiA had suggested that N- and C-terminal residues, directly or indirectly, are involved in the NucA-NuiA interaction (21Meiss G. Gimadutdinow O. Haberland B. Pingoud A. J. Mol. Biol. 2000; 297: 521-534Crossref PubMed Scopus (33) Google Scholar). Nevertheless, the molecular basis for the strong inhibitory interaction has not yet been determined. In comparison with the vast literature on proteinase inhibitors, nuclease inhibitors have received relatively little study. The most detailed investigations have focused on the Bacillus amyloliquefaciens RNase (barnase) inhibitor (barstar) (25Guillet V. Lapthorn A. Hartley R.W. Mauguen Y. Structure (Camb.). 1993; 1: 165-176Abstract Full Text PDF PubMed Scopus (113) Google Scholar), the RNase A inhibitor (26Kobe B. Deisenhofer J. J. Mol. Biol. 1996; 264: 1028-1043Crossref PubMed Scopus (177) Google Scholar), and the immunity proteins that protect Escherichia coli from the colicin DNase activity (27Ko T.P. Liao C.C. Ku W.Y. Chak K.F. Yuan H.S. Structure (Camb.). 1999; 7: 91-102Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 28Kleanthous C. James R. Hemmings A.M. Moore G.R. Biochem. Soc. Trans. 1999; 27: 63-67Crossref PubMed Scopus (7) Google Scholar). Consideration of the structures of these nuclease-inhibitor complexes, as well as the structure of the NucA-NuiA complex determined in the present study, suggests few common modes of inhibition. Here we present the crystal structure of NucA (28 kDa) in complex with NuiA (15 kDa) at a resolution of 2.3 Aå. Many of the features of the NucA-NuiA complex are unique. NuiA interacts directly with residues in the active enzyme site displaying target site mimicry and interacting directly with the active site Mg2+ ion through coordination with the C-terminal Thr-135Nui residue. Binding of NuiA results in no significant change of the backbone atoms of NucA (22Ghosh M. Meiss G. Pingoud A. London R.E. Pedersen L.C. J. Biol. Chem. 2005; 280: 27990-27997Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) but does result in several minor side chain rearrangements. The structure of NucA-complexed NuiA shows some differences relative to the previously determined solution structure (29Kirby T.W. Mueller G.A. DeRose E.F. Lebetkin M.S. Meiss G. Pingoud A. London R.E. J. Mol. Biol. 2002; 320: 771-782Crossref PubMed Scopus (17) Google Scholar) of the uncomplexed inhibitor, primarily in the loop regions. Protein Expression and Purification—The recombinant NucA construct, containing a D121A mutation to reduce activity and related cellular toxicity, lacking the N-terminal export signal peptide, and containing an N-terminal His tag to facilitate purification, was produced as described previously (22Ghosh M. Meiss G. Pingoud A. London R.E. Pedersen L.C. J. Biol. Chem. 2005; 280: 27990-27997Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Recombinant NuiA, also containing an N-terminal His tag, was similarly produced as described previously (29Kirby T.W. Mueller G.A. DeRose E.F. Lebetkin M.S. Meiss G. Pingoud A. London R.E. J. Mol. Biol. 2002; 320: 771-782Crossref PubMed Scopus (17) Google Scholar). E. coli cells containing the required plasmid were grown to mid-log phase (A600 ∼ 0.6) at 37 °C in LB medium containing 30 μg/ml kanamycin. NuiA protein expression was induced with 0.4 mm isopropyl-β-d-thiogalactopyranoside at 37 °C for 6 h. Cells were harvested by centrifugation (at 7000 × g), resuspended in 20 mm Tris-HCl, pH 8.0, 100 mm NaCl, and lysed by sonication with a Branson Sonifier 200 using a Microtip probe. The lysate was centrifuged at 30,000 × g for 40 min. The supernatant was applied to a Ni2+-NTA 2The abbreviations used are: NTA, nitrilotriacetic acid; PDB, Protein Data Bank; PEG, polyethylene glycol; MES, 4-morpholineethanesulfonic acid. resin (Qiagen) equilibrated with extraction buffer and eluted with 20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 200 mm imidazole. The eluted fractions containing NuiA protein were then concentrated using a Millipore concentrator to a desired volume of 10 ml (∼10 mg/ml concentration) and applied to the Superdex-75 gel filtration 2.6 × 60-cm column equilibrated with 20 mm Tris-HCl, pH 8.0, 100 mm NaCl. The protein corresponding to the major absorbance peak at 280 nm was found to be NuiA (∼98% purity) as judged by SDS-polyacrylamide gel electrophoresis. Based on the same criteria, the purity of NucA was estimated to be ∼95%. The N-terminal His tags on both proteins were cleaved by overnight incubation of the protein samples with thrombin (Novagen) at a concentration of 50 units/100 ml at 4 °C. The preparations were then once again passed through Ni2+-NTA resin (Qiagen) equilibrated with 50 mm Tris-HCl, pH 8.0, 200 mm NaCl to remove any residual His-tagged protein as well as the cleaved N-terminal His tag. After the cleavage reaction, the N-terminal residue was glycine, so that the final construct corresponded to NucA Δ1–33, P34G (residues 34–274). Similarly, the NuiA construct that resulted after the cleavage actually corresponded to NuiA M1S (residues 1–135). An additional glycine residue, which theoretically precedes Ser-1 after the thrombin cleavage, was not observed, and hence has not been assigned a position in the construct. The two cleaved proteins were then mixed together and applied to a Superdex-200 gel filtration 2.6 × 60-cm column previously equilibrated with 50 mm Tris-HCl, pH 7.5, 200 mm NaCl, and 2 mm dithiothreitol. The peak corresponding to the NucA-NuiA complex was identified by SDS-polyacrylamide gel electrophoresis. Determination of Inhibition Constants—Values of Ki(app) for the inhibition of NucA by NuiA were determined by measuring the steady-state rate of supercoiled plasmid DNA cleavage in the presence of varying NuiA concentrations using an agarose gel assay. Reactions were performed at 25 °C in a buffer consisting of 50 mm Tris-HCl, pH 7.0, 50 mm NaCl, 1 mm EDTA, 0.01% Triton X-100, 0.01% bovine serum albumin, and5 mm MnCl2 using a concentration of 5 pm NucA and 25 ng/μl plasmid DNA. Values for Ki(app) were calculated by fitting the steady-state rates to Equation 1, which describes tightbinding inhibitors, v=v0[E]-[I]-K+([E]-[I]-K)2+4[E]K2[E]Eq. 1 where ν0 is the steady-state rate of supercoiled plasmid DNA cleavage in the absence of inhibitor, [E] is the active enzyme concentration, [I] is the concentration of the inhibitor, and ν is the steady-state rate in the presence of inhibitor (30Kuzmic P. Elrod K.C. Cregar L.M. Sideris S. Rai R. Janc J.W. Anal. Biochem. 2000; 286: 45-50Crossref PubMed Scopus (47) Google Scholar, 31Williams J.W. Morrison J.F. Methods Enzymol. 1979; 63: 437-467Crossref PubMed Scopus (659) Google Scholar). For mutations other than those involved in phosphate charge mimicry and metal ion bridging, estimates of Ki(app) were calculated according to Equation 2 (30Kuzmic P. Elrod K.C. Cregar L.M. Sideris S. Rai R. Janc J.W. Anal. Biochem. 2000; 286: 45-50Crossref PubMed Scopus (47) Google Scholar) by determining the rate of supercoiled plasmid DNA cleavage by NucA (5 nm) in the absence (v0) and presence (v) of inhibitor (25 nm). K=[I]-[E](1-v/v0)(v0/v-1)Eq. 2 All DNA cleavage reactions were analyzed by electrophoresis on 0.8% agarose gels in Tris borate-EDTA buffer followed by ethidium bromide staining. Crystallization and Data Collection—The NucA-NuiA complex purified by gel filtration chromatography was concentrated to 9 mg/ml and exchanged into 25 mm Tris, pH 7.5, 100 mm NaCl, 2 mm dithiothreitol buffer. Crystals of the protein complex were obtained using the hanging drop vapor diffusion technique at 4 °C by mixing 2 μl of the protein solution with 2 μl of the reservoir solution consisting of 100 mm MES, pH 5.5, and 17–21% PEG 6000. The crystals were transferred to 100 mm MES, pH 5.5, 100 mm NaCl, and 20% PEG 6000 buffer and soaked in 100 mm MES, pH 5.5, 100 mm NaCl, 20% PEG 6000, and 20% ethylene glycol as cryoprotectant. For data collection, crystals were flash-cooled by submersion in liquid nitrogen and placed on the goniometer in a stream of nitrogen gas cooled to -180 °C. A lower resolution data set was collected at 2.9 Aå using a Rigaku 007HF x-ray generator equipped with a Saturn 92 detector. A higher resolution data set was then collected at 2.3 Aå at the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Crystals of the NucA-NuiA complex belong to space group P43212 and contain one molecule each of NucA and NuiA in the asymmetric unit. Structure Determination and Refinement—The crystal structure of NucA (PDB ID code 1ZM8 (22Ghosh M. Meiss G. Pingoud A. London R.E. Pedersen L.C. J. Biol. Chem. 2005; 280: 27990-27997Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar)) was used as the model for molecular replacement using the 2.9-Aå resolution data set. The program Molrep (32Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4148) Google Scholar) from CCP4 (33Bailey S. Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar) was used to calculate phases by molecular replacement. The model for NuiA was built into the electron density following the trace of the NMR structure for NuiA (PDB ID code 1J57 (29Kirby T.W. Mueller G.A. DeRose E.F. Lebetkin M.S. Meiss G. Pingoud A. London R.E. J. Mol. Biol. 2002; 320: 771-782Crossref PubMed Scopus (17) Google Scholar)) that had been placed manually into the electron density. The model was then refined against the 2.3-Aå data set by iterative cycles of model building using the program O (34Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar) and refinement using the program CNS (35Bruönger 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. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). The quality of the final structure was assessed using the programs Procheck (36Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and Molprobity (37Lovell S.C. Davis I.W. Arendall 3rd, W.B. de Bakker P.I. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2003; 50: 437-450Crossref PubMed Scopus (3847) Google Scholar). The final model includes residues 34–274 of NucA and 1–135 of NuiA. The statistics for the data collection and results from refinement are reported in Table 1. The structure of the NucA-NuiA enzyme complex has been submitted to the Protein Data Bank (2O3B).TABLE 1Crystallographic data table for the NucA-NuiA complexCrystallographic data statisticsData setNucA-NuiAUnit cella = b = 87.23; c = 138.98 α = β = γ = 90°Space groupP43212Resolution (Aå)50-2.3No. of observations283,171Unique reflections24,604Rsym (%) (last shell)aRsym = Σ(|Ii – 〈I 〉|)/Σ(Ii) where Ii is the intensity of the ith observation and 〈I 〉 is the mean intensity of the reflection10.0 (57.3)I/σI (last shell)9.4 (3.0)Mosaicity0.23–0.77Completeness (%) (last shell)99.7 (97.5)Refinement statisticsRcryst (%)bRcryst = Σ||Fo| – |Fc||/Σ|Fo| calculated from working data set20.1Rfree (%)cRfree was calculated from 5% of data randomly chosen not to be included in refinement24.1No. of waters229Mean B value (Aå)46.9r.m.s.d.dr.m.s.d., root mean square deviation from ideal valuesBond length (Aå)0.008Bond angle (°)1.3Dihedral angle (°)24.0Improper angle (°)0.82Ramachandran statisticseRamachandran results were determined by using MolProbityResidues in favored (98%) regions (%)95.8Residues in allowed (>99.8%) regions (%)100a Rsym = Σ(|Ii – 〈I 〉|)/Σ(Ii) where Ii is the intensity of the ith observation and 〈I 〉 is the mean intensity of the reflectionb Rcryst = Σ||Fo| – |Fc||/Σ|Fo| calculated from working data setc Rfree was calculated from 5% of data randomly chosen not to be included in refinementd r.m.s.d., root mean square deviatione Ramachandran results were determined by using MolProbity Open table in a new tab Crystal Structure of the NucA-NuiA Complex—The crystal structure of the NucA-NuiA complex was determined at 2.3-Aå resolution using recombinant NucA and NuiA expressed in E. coli (Table 1). A ribbon model representing the secondary structure of the complex is shown in Fig. 1A. As seen in the previously determined structure of NucA (22Ghosh M. Meiss G. Pingoud A. London R.E. Pedersen L.C. J. Biol. Chem. 2005; 280: 27990-27997Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), the secondary structure is composed of 13 α-helices and two β-sheets. The root mean square deviation between the complexed and free NucA structure (Fig. 1B) is 0.34 Aå for all Cα atoms. A divalent metal ion is located in the active site of NucA, and two additional divalent metal ions were observed in the secondary metal ion-binding site previously identified in the NucA structure (22Ghosh M. Meiss G. Pingoud A. London R.E. Pedersen L.C. J. Biol. Chem. 2005; 280: 27990-27997Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The coordination geometry of the active site metal ion is consistent with Mg2+, the endogenous catalytic metal. Alternatively, the identities of the two metal ions at the secondary site were found to be most consistent with Ni2+ ions. These were presumably acquired or exchanged into the NucA molecule as a result of passage through the Ni2+-NTA column (38Hannan J.P. Whittaker S.B. Davy S.L. Kuhlmann U.C. Pommer A.J. Hemmings A.M. James R. Kleanthous C. Moore G.R. Protein Sci. 1999; 8: 1711-1713Crossref PubMed Scopus (11) Google Scholar). Consistent with the previously determined solution structure (29Kirby T.W. Mueller G.A. DeRose E.F. Lebetkin M.S. Meiss G. Pingoud A. London R.E. J. Mol. Biol. 2002; 320: 771-782Crossref PubMed Scopus (17) Google Scholar), NuiA (Fig. 1B) consists of four helices and a central six-stranded β-sheet arranged as an αβα sandwich. Helices A, C, and D are positioned on one side of the central β-sheet, and the distorted helix B is positioned on the other side. The three central strands of the β-sheet are located at the C terminus of the protein, and all six of the strands are antiparallel. Omitting the poorly aligned region at the end of helix 4 of NuiA (residues 71–90), the root mean square deviation for the backbone atoms of the NuiA structure determined in solution relative to that in the crystallized NucA-NuiA complex was 3.46 Aå. We note that a more recent structural calculation using the same data set in combination with the program CYANA (39Guntert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2553) Google Scholar, 40Herrmann T. Guntert P. Wuthrich K. J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1327) Google Scholar) resulted in much closer agreement, with the root mean square deviation for the full structure falling to 3.1 Aå. 3E. F. DeRose, T. W. Kirby, G. A. Mueller, and R. E. London, unpublished results. In the previously determined structure of NucA, the secondary metal ion-binding site was found to constitute a lattice contact, with two carboxyl oxygen atoms of a Glu-136Nuc residue on a symmetry-related NucA molecule making contact with both metal ions in the secondary metal ion-binding site (22Ghosh M. Meiss G. Pingoud A. London R.E. Pedersen L.C. J. Biol. Chem. 2005; 280: 27990-27997Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In the structure of the NucA-NuiA complex, there is also a lattice contact involving the secondary metal ion-binding site on NucA and the Asp-87Nui residue of a second, symmetry-related NuiA molecule. The NucA-NuiA Interface—The structure of NuiA can be described as an “open jaw” biting into one side of the NucA molecule in the complex (Fig. 1, C and D). The Thr-135Nui and Glu-24Nui residues of the upper jaw enter the NucA active site, whereas Asp-75Nui and Trp-76Nui residues of the lower jaw engulf a protruding section of the nuclease that includes a long loop of NucA running from Arg-93Nuc into the beginning of β-strand 4 (Arg-122Nuc) (Fig. 2A). Close contact is also made with several residues on coaxial helices H (Thr-151Nuc–Asn-155Nuc) and I (Thr-158Nuc–Gln-172Nuc) of NucA, including Asn-155Nuc and Glu-163Nuc (Fig. 2, B and C). The major hydrogen-bonding and salt bridge interactions between NucA and NuiA are summarized in Table 2 and illustrated in Fig. 2A. These include salt bridge interactions between Arg-93Nuc and Glu-24Nui, Arg-93Nuc and the C-terminal Thr-135Nui carboxyl oxygen, and Lys-101Nuc and Asp-75Nui in addition to a network of direct and water-mediated hydrogen-bonding interactions. The solvent-accessible surface areas calculated individually are 10,200 Aå2 for NucA and 7488 Aå2 for NuiA. After complex formation, the buried solvent-accessible surface area at the interface is calculated to be 1391 Aå2.TABLE 2Hydrogen-bonding interaction between NucA and NuiA Acceptor-donor distances are based on ≤3.4 Aå.NucA residuesDistanceWaterDistanceNuiA residuesAåAåArg-93 NH23.0Glu-24 OE2Arg-93 NH13.1Thr-135 OGln-94 O3.3Arg-69 NELys-101 NZ3.1Asp-75 OD2Ser-113 OG3.3Glu-80 OE1Ser-113 OG3.4Glu-81 OE2Gly-117 N3.2Glu-26 OE1Arg-122 NH13.0Ser-23 OArg-122 N2.8Glu-24 OAsn-155 ND12.8Glu-24 OE1Asn-155 OD12.9Thr-135 OG1Glu163 OE13.4Thr-135 OXTMG2+2.0Thr135 OG1Arg-122 O3.0W13.0Thr-135 OG1His-124 ND12.8W1Asn-155 OD13.0W1W13.0Glu-24 OE1Ser-128 N3.0W382.6Thr-135 OGlu-163 OE12.6W862.9Thr-135 OG1Asn-155 O2.8W38Glu-163 OE22.7W873.3Thr-135 OG1MG2+2.3W87Ala-129 N2.9W1652.7Thr-135 OXT2.8Thr-135 OArg-167 NE23.0W1662.4Thr-135 OXTGlu-163 OE12.5W166Arg-122 NH13.2W1962.7Glu-26 OE1 Open table in a new tab The substantial electrostatic contribution to the NucA-NuiA interaction is illustrated by the GRASP-generated surfaces in Fig. 3. To reveal the electrostatic potential of the interface, NucA is represented by its electrostatic surface, whereas NuiA is represented by a ribbon diagram (Fig. 3A). The representation is reversd in Fig. 3B. The electrostatic representations in Fig. 3 are consistent with the entries in Table 2, which include one acidic and five basic residues for NucA and one basic and seven acidic residues for NuiA. This electrostatic pattern is consistent with the proposal that, to a significant extent, NuiA binds to NucA as a substrate mimic. The principal hydrophobic contributions to the interface include Phe-97Nuc–Glu-24Nui (methylenes), Pro-99Nuc–Trp-76Nui, and several other side chain interactions. However, there is no concentration of hydrophobic residues in the interface. The structure of the complex also contains two bound MES molecules, one of which contacts both NucA and NuiA. Its primary contacts include Glu-92Nuc and Arg-106Nui. Effect of NuiA Interface Mutations—A previous deletion mutant of NuiA demonstrated the importance of the C terminus for tight binding to NucA (21Meiss G. Gimadutdinow O. Haberland B. Pingoud A. J. Mol. Biol. 2000; 297: 521-534Crossref PubMed Scopus (33) Google Scholar). Our structural analysis confirms a critical role for Thr-135 of NuiA in NucA binding and identifies this residue as being involved in a novel metal ion bridge as part of the NucA-NuiA interface between the C-terminal end of NuiA and the NucA active site. Deletion of the C-terminal residue of NuiA (NuiA-Δ135) resulted in a 600-fold increase in the inhibition constant (Ki) relative to the value for the wild type inhibitor (Table 3). This result indicates the significance of metal ion bridging for tight binding of NuiA to NucA. In addition, our structural analysis indicates that Glu-24Nui is inserted into the active site of NucA, apparently mimicking the negative charge of the scissile phosphate. To obtain quantitative data on the contribution of this residue to the inhibition of NucA by NuiA we generated three mutants of the inhibitor with a conservative (Asp), an isosteric (Gln), and a nonconservative (Ala) amino acid substitution at this position and determined the Ki values for these variants (Table 3). The Ki values obtained show the greatest increase (about 4 orders of magnitude) for the nonconservative and the isosteric substitutions (variants E24A and E24Q), whereas a wild type-like Ki was determined for variant E24D. Thus, a conservative amino acid exchange that preserves a negative ch" @default.
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• W2133187258 title "The Nuclease A-Inhibitor Complex Is Characterized by a Novel Metal Ion Bridge" @default.
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