NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis
2005; Elsevier BV; Volume: 280; Issue: 34 Linguagem: Inglês
10.1074/jbc.m503780200
ISSN1083-351X
AutoresSandeep Kumar Srivastava, Rama P. Tripathi, Ravishankar Ramachandran,
Tópico(s)HIV/AIDS drug development and treatment
ResumoDNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents. DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents. DNA ligases are vital enzymes in replication and repair and catalyze the formation of a phosphodiester linkage between adjacent termini in double-stranded DNA through similar mechanisms (1Lehman I.R. Science. 1974; 186: 790-797Crossref PubMed Scopus (472) Google Scholar). These enzymes can be divided into two classes, viz. NAD+- and ATP-dependent ligases, based on the cofactor specificities (2Engler M.J. Richardson C.C. Boyer P.D The Enzymes. 15. Academic Press, New York1982: 3-29Google Scholar). NAD+-dependent DNA ligases, commonly called LigA, are found in bacteria and entomopox-viruses (3Wilkinson A. Day J. Bowater R. Mol. Microbiol. 2001; 40: 1241-1248Crossref PubMed Scopus (182) Google Scholar, 20Sriskanda V. Moyer R.W. Shuman S. J. Biol. Chem. 2001; 276: 36100-36109Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), whereas ATP-dependent ligases are ubiquitous (3Wilkinson A. Day J. Bowater R. Mol. Microbiol. 2001; 40: 1241-1248Crossref PubMed Scopus (182) Google Scholar). Although there is little sequence homology between the eubacterial and eukaryotic enzymes, they exhibit some structural homology in specific domains (4Timson D.J. Singleton M.R. Wigley D.B. Mutat. Res. 2000; 460: 301-318Crossref PubMed Scopus (130) Google Scholar, 5Doherty A.J. Suh S.W. Nucleic Acids Res. 2000; 28: 4051-4058Crossref PubMed Scopus (132) Google Scholar). The mechanistic steps involved in enzymatic action are also broadly conserved. Briefly, in the first step, the mode of action involves an attack on the α-phosphorus of ATP or NAD+ by the enzyme, releasing pyrophosphate or NMN and forming a ligase-adenylate intermediate. In the second step, the bound AMP is transferred to the 5′-end of DNA to form a DNA-adenylate intermediate. AMP is released in the third step, where the protein catalyzes the joining of the 3′-nicked DNA to the DNA-adenylate intermediate. These steps involve large conformational changes and also encircling and partial unwinding of the nicked DNA substrate (6Pascal J.M. O'Brien P.J. Tomkinson A.E. Ellenberger T. Nature. 2004; 432: 473-478Crossref PubMed Scopus (264) Google Scholar, 7Gajiwala K.C. Pinko C. Structure (Camb). 2004; 12: 1449-1459Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 8Lee J.Y. Chang C. Song H.K. Moon J. Yang J.K. Kim H.-K. Kwon S.T. Suh S.W. EMBO J. 2000; 19: 1119-1129Crossref PubMed Scopus (154) Google Scholar). Some bacteria code for both NAD+- and ATP-dependent DNA ligases (3Wilkinson A. Day J. Bowater R. Mol. Microbiol. 2001; 40: 1241-1248Crossref PubMed Scopus (182) Google Scholar, 9Cheng C. Shuman S. Nucleic Acids Res. 1997; 25: 1369-1374Crossref PubMed Scopus (47) Google Scholar). Mycobacterium tuberculosis codes for at least three different types of ATP-dependent ligases and a NAD+-dependent ligase (10Gong C. Martins A. Bongiorno P. Glickman M. Shuman S. J. Biol. Chem. 2004; 279: 20594-20606Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 11Wilkinson A. Sayer H. Bullard D. Smith A. Day J. Kieser T. Bowater R.P. Proteins Struct. Funct. Genet. 2003; 51: 321-326Crossref PubMed Scopus (20) Google Scholar). Gene knockout and other studies have shown LigA to be indispensable in several bacteria, including Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and M. tuberculosis (10Gong C. Martins A. Bongiorno P. Glickman M. Shuman S. J. Biol. Chem. 2004; 279: 20594-20606Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 12Dermody J.J. Robinson G.T. Sternglanz R. J. Bacteriol. 1979; 139: 701-704Crossref PubMed Google Scholar, 13Kaczmarek F.S. Zaniewski R.P. Gootz T.D. Danley D.E. Mansour M.N. Griffor M. Kamath A.V. Cronan M. Mueller J. Sun D. Martin P.K. Benton B. McDowell L. Biek D. Schmid M.B. J. Bacteriol. 2001; 183: 3016-3024Crossref PubMed Scopus (48) Google Scholar, 14Petit M.A. Ehrlich S.D. Nucleic Acids Res. 2000; 28: 4642-4648Crossref PubMed Scopus (48) Google Scholar, 15Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (2018) Google Scholar). No LigA structure from mycobacterial sources is available to date. However, the crystal structure of the full-length protein is available for the Thermus filiformis enzyme (TfiLigA), 1The abbreviations used are: TfiLigA, T. filiformis LigA; EfaLigA, E. faecalis LigA; MtuLigA, M. tuberculosis LigA; MICs, minimum inhibitory concentrations; EcoLigA, E. coli LigA. whereas structures of the adenylation domain are available for the Bacillus stearothermophilus and Enterococcus faecalis (EfaLigA) enzymes (7Gajiwala K.C. Pinko C. Structure (Camb). 2004; 12: 1449-1459Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 8Lee J.Y. Chang C. Song H.K. Moon J. Yang J.K. Kim H.-K. Kwon S.T. Suh S.W. EMBO J. 2000; 19: 1119-1129Crossref PubMed Scopus (154) Google Scholar, 16Singleton M.R. Hakansson K. Timson D.J. Wigley D.B. Structure (Camb.). 1999; 7: 35-42Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The structures have shown that the enzyme has a modular architecture consisting of distinct domains. The adenylation domain contains the cofactor-binding site and can be divided further into two subdomains. Subdomain 1a contains the NMN-binding pocket, whereas subdomain 1b contains the AMP-binding site. The EfaLigA structures show that the NAD+-binding site is generated by a specific spatial disposition of the two subdomains where subdomain 1a is in close proximity to the AMP-binding site in subdomain 1b (7Gajiwala K.C. Pinko C. Structure (Camb). 2004; 12: 1449-1459Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Structure-based mutagenesis experiments have also led to the identification of residues important for NAD+ recognition and support systematic active-site remodeling in different reaction steps (17Zhu H. Shuman S. J. Biol. Chem. 2005; 280: 12137-12144Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). With the problem of multiple drug resistance spreading across the world, it is important to find inhibitors from different chemical classes with new modes of action. In this context, specific inhibitors for NAD+-dependent ligases are being identified, as no drug is known to act against this enzyme so far. Other groups have very recently identified compounds belonging to arylamino and pyridochromanone classes as specific inhibitors of NAD+-dependent DNA ligases (18Ciarrocchi G. MacPhee D.G. Deady L.W. Tilley L. Antimicrob. Agents Chemother. 1999; 43: 2766-2772Crossref PubMed Google Scholar, 19Brőtz-Oesterhelt H. Knezevic I. Bartel S. Lampe T. Warnecke-Eberz U. Ziegelbauer K. Häbich D. Labischiinski H. J. Biol. Chem. 2003; 278: 39435-39442Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). New inhibitors can also be potentially used as broad bactericidal agents, as NAD+-specific enzymes have not been identified in eukaryotic genomes and are exclusively found in eubacteria (3Wilkinson A. Day J. Bowater R. Mol. Microbiol. 2001; 40: 1241-1248Crossref PubMed Scopus (182) Google Scholar) and some viruses (20Sriskanda V. Moyer R.W. Shuman S. J. Biol. Chem. 2001; 276: 36100-36109Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In this work, we report the crystal structure of the adenylation domain of the M. tuberculosis NAD+-dependent DNA ligase (MtuLigA) bound to AMP. The structure captures a new spatial disposition of the two subdomains in the protein. The AMP conformation in the crystal structure is different from that observed in the TfiLigA structure, but is similar to the AMP part of NAD+ in its co-crystal structure with Efa-LigA. Based on the crystal structure and in silico docking studies, we have identified glycosyl ureides as a new class of DNA ligase inhibitors. In vitro assays and bactericidal activities assayed using specific E. coli and Salmonella typhimurium strains demonstrated that the compounds are able to distinguish between NAD+- and ATP-dependent ligases. Although the M. tuberculosis enzyme was inhibited in the low micromolar range, human DNA ligase I was inhibited only at much higher concentrations. Cloning, Expression, and Purification—The adenylation domain of MtuLigA (Rv3014c) consists of residues 1-328. The DNA sequence encoding this domain was PCR-amplified from genomic DNA of M. tuberculosis H37Rv using forward primer 5′-GGAATTCCATGGGCTCCCCAGACGCCG-3′ and reverse primer 5′-ATCGGATCCCTCGGGCGGGTACTTGTAGG-3′ containing NcoI and BamHI restriction sites (underlined), respectively. The amplified PCR product was digested and ligated into pQE60 (Qiagen Inc.) digested at same site. Incorporation of NcoI into the forward primer leads to replacement of the first two amino acids in the sequence: valine and serine to methionine and glycine, respectively. The integrity of the insert was verified by sequencing. The construct was transformed into E. coli BL21(DE3) cells (Novagen) and grown in LB medium containing 0.1 mg/ml ampicillin to A600 ∼ 0.5. Protein expression was induced by addition of 0.8 mm isopropyl β-d-thiogalactopyranoside at 28 °C for 8 h. Cells were harvested by centrifugation; resuspended in 50 mm Tris-HCl (pH 8.0), 200 mm NaCl, and 10 mm imidazole (buffer A); and lysed by sonication. The crude lysate was centrifuged at 27,000 × g for 30 min. The supernatant was applied to a nickel-iminodiacetic acid column (Amersham Biosciences) equilibrated with buffer A, and protein was eluted using a 10-500 mm imidazole gradient. Purified fractions were pooled, precipitated using ammonium sulfate (45% saturation), redissolved in a minimum volume of buffer B (50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 1 mm EDTA, and 2 mm dithiothreitol), and loaded onto a Superdex S-200 gel filtration column (Amersham Biosciences) equilibrated with buffer B. Purified protein was pooled and concentrated to 15 mg/ml using a Centricon concentrator (10-kDa cutoff; Amicon, Inc.). Protein concentrations were determined with Bradford reagent (21Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217546) Google Scholar) using bovine serum albumin as a standard. Crystallization and Data Collection—Crystals of the MtuLigA adenylation domain were grown by vapor diffusion using the hanging drop method. A drop containing 2 μl each of 12 mg/ml protein solution in buffer B containing 4 mm NAD+ and reservoir solution containing 0.1 m NaCl, 0.1 m Na-HEPES (pH 7.6), and 1.5 m (NH4)2SO4 kept for 1 week at 24 °C yielded crystals of typical dimensions (0.7 × 0.5 × 0.2 mm). These were mounted on capillaries, and x-ray data were collected at room temperature on a MAR imaging plate mounted on a Rigaku rotating anode generator. The crystals diffracted weakly to 3.15 Å, and the data were overall complete to 99.4% with an average redundancy of 8.7. Data integration, reduction, and scaling were performed using the DENZO/SCALEPACK suite of programs (22Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The data collection statistics are summarized in Table I.Table IData collection and refinement statistics Values in parentheses are for the highest resolution bin (3.26 to 3.15 Å).Data collectionSpace groupP6122Cell dimensions (Å)a = b = 95.81, c = 208.39Wavelength (Å)1.5418Resolution (Å)3.15No. of measured reflections89,977No. of unique reflections10,337Average multiplicity8.7Average I/σ(I)4.4Completeness (%)99.4 (99.6)Rmerge (%)15.3 (89.1)Rrim (%)aRrim = Σhkl (N/(N–1))Σ1I1 (hkl)–I(hkl)/Σhkl/Σ1I1(I1(hkl)16.3 (94.1)RefinementRefinement limits (Å)15 to 3.15No. of protein atoms2271No. of cofactor atoms22No. of water oxygens4Rcryst (%)25.3Rfree (%)31.4r.m.s.d.bRoot mean square deviation in bond lengths (Å)0.005r.m.s.d. in bond angles1.1°r.m.s.d. in dihedrals26.7°a Rrim = Σhkl (N/(N–1))Σ1I1 (hkl)–I(hkl)/Σhkl/Σ1I1(I1(hkl)b Root mean square deviation Open table in a new tab Structure Solution and Refinement—The structure was solved using CCP4 (23Collaborative Computing Project No. 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) and AMoRE (24Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) with models derived from the T. filiformis structure (Protein Data Bank code 1V9P) after stripping it of cofactors and solvent molecules. The two subdomains of the adenylation domain were placed independently in the asymmetric unit. Refinements were carried out using XPLOR (25Brünger A.T. Karplus M. Petsko G.A. Acta Crystallogr. Sect. A. 1989; 45: 50-61Crossref Scopus (272) Google Scholar), whereas model building was carried out using Turbo-Frodo (26Roussel A. Cambillau C. Silicon Graphics Geometry Partner Directory. Silicon Graphics, Inc., Mountain View, CA1989: 77-78Google Scholar). Rounds of model building and simulated annealing refinement were continued until R and Rfree values converged to 25.3 and 31.4%, respectively. The final model consists of residues 8-328, AMP, and 4 water molecules. More than 91% of all residues are in the core regions of the Ramachandran map (27Ramachandran G.N. Sasisekharan V. Adv. Protein Chem. 1968; 23: 283-438Crossref PubMed Scopus (2770) Google Scholar). The geometric parameters are also well within acceptable values for a model at this resolution. The refinement and model statistics are summarized in Table I. The coordinates have been submitted to the Protein Data Bank with code 1ZAU (28Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27937) Google Scholar). In Silico Docking—The current crystal structure and also that of NAD+-bound EfaLigA (Protein Data Bank code 1TAE) were used as models in in silico ligand docking calculations using the programs AutoDock Version 3.0 (29Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9301) Google Scholar) and Gold Version 2.2 (30Verdonk M.L. Cole J.C. Hartshorn M.J. Murray C.W. Taylor R.D. Proteins. 2003; 52: 609-623Crossref PubMed Scopus (2298) Google Scholar). We used a Perl/Python-based script to add the capability of automated docking against a ligand data base to AutoDock. A computer cluster consisting of Silicon Graphics Origin 350 servers and Silicon Graphics Octane workstations was used for the computation and analysis of docked complexes. The NAD+-binding site in MtuLigA was generated by superposing subdomain 1a onto the orientation observed in the NAD+-bound structure of EfaLigA (Protein Data Bank code 1TAE). The docked ligands form part of an in-house collection of ∼15,000 compounds whose synthesis expertise is also available. It can be filtered for activity against tuberculosis, etc., based on prior in-house experiments. Control docking runs to optimize the docking parameters were carried out using AMP and NAD+, whose cocrystal structures with LigA were available in the Protein Data Bank for comparison. Selected compounds from the best 10% docked complexes (as observed from the AutoDock scoring and Gold fitness scores) were taken up further for in vitro and in vivo ligase assays. In Vitro Activity—In vitro assays for ligase activity were performed using a 40-bp double-stranded DNA substrate carrying a single-strand nick between bases 22 and 23 (31Timson D.J. Wigley D.B. J. Mol. Biol. 1999; 285: 73-83Crossref PubMed Scopus (66) Google Scholar). This substrate was created in Tris/EDTA buffer by annealing a 22-mer (5′-CCT GGA CAT AGA CTC GTA CCT T-3′) and a 18-mer (5′-AGC TGG ATC ACT GGA CAT-3′) to a complementary 40-mer (5′-ATG TCC AGT GAT CCA GCT AAG GTA CGA GTC TAT GTC CAG G-3′). The 18-mer was radiolabeled at the 5′-end by incubating 10 μg of the oligonucleotide with 100 μCi of [γ-32P]ATP (3000 Ci/mmol; Board of Radiation and Isotope Technology) and 30 units of T4 polynucleotide kinase for 1 h, followed by 10 min at 70 °C (32Doherty A.J. Ashford S.R. Subramanya H.S. Wigley D.B. J. Biol. Chem. 1996; 271: 11083-11089Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The unincorporated label was removed using a Sephadex G-25 column. The labeled 40-bp nicked DNA substrate was used to assay the in vitro inhibitory activity of different compounds against MtuLigA, bacteriophage T4 ligase, and human DNA ligase I. The full-length MtuLigA protein was cloned into the NdeI/NcoI-digested pET41a vector (Novagen). After expression in E. coli BL21(DE3) cells, the C-terminally His-tagged protein was purified according to standard procedures. The assays were done with 2 ng of the purified protein. Reaction mixtures (15 μl) containing 50 mm Tris-HCl (pH 8.0), 5 mm dithiothreitol, 10 mm MgCl2, 10% Me2SO, 2 μm NAD+, 2 pmol of 32P-labeled nicked duplex DNA substrate, and different concentration of compounds were incubated for 1 h at 25 °C. Reactions were quenched with formamide and EDTA. The reaction products were resolved electrophoretically on 15% polyacrylamide gel containing 8 m urea in 90 mm Tris borate and 2.5 mm EDTA. Autoradiograms of the gels were developed, and the extent of ligation was measured by scanning the gel using ImageMaster 1D Elite software (Amersham Biosciences). All the compounds were dissolved in 100% Me2SO. The compound solutions comprised 0.1 volume of the ligation reaction mixture; thus, 10% Me2SO was included in all the control reactions. The activity assay was performed in the same way for T4 ligase in a volume of 15 μl containing 0.05 unit of enzyme (Amersham Biosciences), 2 pmol of labeled template, and 66 μm ATP in 66 mm Tris-HCl (pH 7.6), 6.6 mm MgCl2,10mm dithiothreitol, and 10% Me2SO. The human DNA ligase I expression plasmid was transformed into E. coli BL21(DE3) cells and purified as described previously (33Mackenney V.J. Barnes D.E. Lindahl T. J. Biol. Chem. 1997; 272: 11550-11556Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Purified protein was concentrated to 2 mg/ml. 2 μg protein was used for assay in 50 mm Tris-HCl (pH 8.0), 10 mm MgCl2,5mm dithiothreitol, 50 μg/ml bovine serum albumin, and 1 mm ATP as described above. The IC50 values were determined by plotting the relative ligation activity versus inhibitor concentration and fitting to equation Vi/V0 = IC50/(IC50 + [I]) using GraphPad Prism®. V0 and Vi represent the rates of ligation in the absence and presence of inhibitor, respectively, and [I] refers to the inhibitor concentration. Antibacterial Activity and Inhibition of Ligase in Vivo—The recombinant plasmid pRBL (34Ren Z.J. Baumann R.G. Black L.W. Gene (Amst.). 1997; 195: 303-311Crossref PubMed Scopus (29) Google Scholar) containing the gene for T4 DNA ligase in pTrc99A was transformed into the E. coli GR501 ligAts mutant (35Lavesa-Curto L. Sayer H. Bullard D. MacDonald A. Wilkinson A. Smith A. Bowater L. Hemmings A. Bowater R.P. Microbiology. 2004; 150: 4171-4180Crossref PubMed Scopus (21) Google Scholar). To have the same genetic background, the M. tuberculosis ligA gene was amplified from genomic DNA using primers containing sites for NcoI and HindIII, cloned into NcoI- and HindIII-digested pTrc99A (36Amann E. Ochs B. Abel K.J. Gene (Amst.). 1988; 69: 301-315Crossref PubMed Scopus (881) Google Scholar), and transformed into E. coli GR501. In growth experiments, the strains expressing MtuLigA or T4 DNA ligase were compared with a control GR501 strain carrying empty pTrc99A without any gene insertions at 37 °C. As reported previously (19Brőtz-Oesterhelt H. Knezevic I. Bartel S. Lampe T. Warnecke-Eberz U. Ziegelbauer K. Häbich D. Labischiinski H. J. Biol. Chem. 2003; 278: 39435-39442Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and reproduced by us, the E. coli GR501 ligAts strain grows well at 30 °C, whereas it is strongly delayed at 37 °C. Complementation with either MtuLigA or T4 ligase restores the growth of the mutant strain. Minimum inhibitory concentrations (MICs) of the inhibitors were determined for MtuLigA and T4 DNA ligase in the E. coli GR501 ligAts mutant and in S. typhimurium LT2 (37Park U.E. Olivera B.M. Hughes K.T. Roth J.R. Hillyard D.R. J. Bacteriol. 1989; 171: 2173-2180Crossref PubMed Google Scholar) and its DNA ligase-null mutant derivative, which had been rescued with a plasmid (pBR313/598/8/1b) encoding the T4 DNA ligase gene (38Wilson G.G. Murray N.E. J. Mol. Biol. 1979; 132: 471-491Crossref PubMed Scopus (65) Google Scholar), to check the specificity of compounds for NAD+-dependent ligases from other sources as well. Antimicrobial activity was monitored in microtiter plates using a microdilution assay technique in a volume of 200 μl. Approximately 105 colony-forming units/ml in the case of the E. coli ligAts mutant and 106 colony-forming units/ml in the case of S. typhimurium LT2 and its LigA- mutant strain, rescued with T4 DNA ligase, were incubated with different compound concentrations under ambient conditions for 20 h, and MICs were determined on the basis of the presence of any visible growth. The E. coli mutant strain was grown in LB medium, whereas the S. typhimurium strains were grown in nutrient broth. The media contained 20 μg/ml polymyxin B nonapeptide to facilitate passage of the inhibitors across the outer membrane. Growth Inhibition Studies—To investigate the specificity and sensitivity of the compounds to NAD+-dependent ligase, exponentially growing cultures of S. typhimurium LT2 and its DNA ligase-null mutant derivative in nutrient broth were treated at A600 = 0.4 with increasing compound concentrations. The effect on the growth and viability of both the strains was compared by monitoring A600 and the number of colony-forming units for 4-5 h after addition of the compound. Serially diluted culture aliquots of both strains in phosphate-buffered saline were plated on nutrient agar, and visible colonies were counted after incubating the plates for 15 h at 37 °C. DNA-Inhibitor Interaction—In this assay, the DNA intercalating properties of the inhibitors were measured by the ability to compete with ethidium bromide for DNA binding. Detection of ethidium bromide displacement from DNA, if any, is based on the strong loss of fluorescence that should occur upon its detachment from DNA (39Le Pecq J.-B. Paoletti C. J. Mol. Biol. 1967; 27: 87-106Crossref PubMed Scopus (2186) Google Scholar). The assay mixture contained, in a volume of 100 μl, 5 μg of calf thymus DNA, 5 μm ethidium bromide, 25 mm Tris-HCl (pH 8.0), 50 mm NaCl, and 1 mm EDTA. Upon addition of the inhibitor at increasing concentrations, ethidium bromide fluorescence was immediately detected at an excitation wavelength of 485 nm and an emission wavelength of 612 nm. The adenylation domain of LigA contains all the residues necessary for AMP/NAD+ binding. This domain (consisting of residues 1-328 in MtuLigA) was cloned, expressed, and purified as described. The adenylation domain itself consists of two subdomains. Subdomain 1a is known to be flexible and adopts different spatial dispositions relative to subdomain 1b (7Gajiwala K.C. Pinko C. Structure (Camb). 2004; 12: 1449-1459Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 8Lee J.Y. Chang C. Song H.K. Moon J. Yang J.K. Kim H.-K. Kwon S.T. Suh S.W. EMBO J. 2000; 19: 1119-1129Crossref PubMed Scopus (154) Google Scholar). We therefore carried out independent molecular replacement calculations for the subdomains. The cofactor was not used in the calculations (Fig. 1). Clear connectivity was observed in the initial electron density maps between the two subdomains. We added NAD+ under the crystallization conditions, but well defined density only for noncovalently bound AMP was observed in the initial electron density map itself (Fig. 2). The data collection and refinement statistics are summarized in Table I.Fig. 2An initial 2Fo - Fc electron density map calculated after molecular replacement at 3.15 Å contoured at 1.2σ around the AMP-binding site. The cofactor was not included in the calculations. The refined coordinates are superposed onto the map. AMP (pink) and interacting residues (violet) are labeled for clarity. Polar interactions within 3.5 Å are also indicated by dotted lines. The figure was made using Turbo-Frodo.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Subdomain 1a consists of residues 1-76 and contains residues involved in NMN recognition. This subdomain consists mainly of two helical stretches (Figs. 1 and Fig. 3A). Subdomain 1b contains bound AMP in the crystal structure and consists of residues 77-328. The two domains adopt a novel relative spatial disposition in the structure (Fig. 3). Subdomain 1a in the B. stearothermophilus LigA structure is at one end of the conformation spectrum, whereas in TfiLigA, it is at the other end (Fig. 3B). In EfaLigA, this domain comes in close proximity to the AMP-binding site and generates the complete NAD+-binding site. This subdomain in MtuLigA adopts a new spatial disposition between the above two extremes. Adenylation Site and AMP Conformation—The adenylation domain contains five of six conserved sequence motifs in NAD+ ligases (40Shuman S. Schwer B. Mol. Microbiol. 1995; 17: 405-410Crossref PubMed Scopus (190) Google Scholar). These mainly line the AMP/NAD+-binding pocket. The active-site lysine (Lys123 in MtuLigA), which covalently binds AMP to form the ligase-adenylate intermediate in the first step of the reaction, is part of the conserved motif I, whereas a Glu residue (Glu184 in MtuLigA), which apparently discriminates between AMP conformations, is part of motif III (17Zhu H. Shuman S. J. Biol. Chem. 2005; 280: 12137-12144Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Intriguingly, in the present structure, AMP is not observed to form the phosphoamide adduct with the motif I lysine. In fact, the side chain of this residue appears to be more mobile as evidenced by weaker electron density. The only other AMP-bound LigA structure is that of TfiLigA, whereas an NAD+-bound structure is available for EfaLigA. Although the model used for molecular replacement calculations was derived from TfiLigA, the AMP molecule adopts a conformation similar to that of the AMP part in the NAD+-bound EfaLigA structure (Protein Data Bank code 1TAE). In the TfiLigA structure, the adenylation domain is in an "open" state (Fig. 3B), and the adenosine nucleoside moiety of the covalently bound AMP adopts an anti-conformation. In the NAD+-bound EfaLigA structure, the adenylation domain is observed to be in a "closed" state, and the adenosine nucleoside moiety adopts a syn-conformation (41Shuman S. Lima C. Curr. Opin. Struct. Biol. 2004; 14: 757-764Crossref PubMed Scopus (157) Google Scholar). In the present structure, although the adenylation domain adopts an open conformation, the adenosine nucleoside moiety adopts a syn-conformation. Residues that are <4 Å from the AMP moiety in MtuLigA are Leu90, Ser91, Leu92, Asn94, Glu121, Leu122, Lys123, Ala124, Ala128, Arg144, Glu184, His236, Val298, and Lys300. The possible hydrogen bonds with AMP are indicated in Fig. 2. Lys123 is the residue that should covalently bind to AMP to form the adenylate intermediate, although it is not covalently bound in
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