Structure of the Thiostrepton Resistance Methyltransferase·S-Adenosyl-l-methionine Complex and Its Interaction with Ribosomal RNA

The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)·S-adenosyl-l-methionine (AdoMet) complex was determined at 2.45-Å resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal “L30-like” putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr·rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme. The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)·S-adenosyl-l-methionine (AdoMet) complex was determined at 2.45-Å resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal “L30-like” putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr·rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme. RNA modifications and the enzymes that catalyze their formation are critical for cellular viability. Certain RNA modifications are extremely well characterized, such as CCA addition and amino acylation of the 3′-ends of tRNA, and the contributions of some nucleotide modifications to the creation of specific functional tRNA structures (1Gustilo E.M. Vendeix F.A. Agris P.F. Curr. Opin. Microbiol. 2008; 11: 134-140Crossref PubMed Scopus (180) Google Scholar, 2Clouet-d'Orval B. Gaspin C. Mougin A. Biochimie (Paris). 2005; 87: 889-895Crossref PubMed Scopus (33) Google Scholar, 3Ferré-D'Amaré A.R. Curr. Opin. Struct. Biol. 2003; 13: 49-55Crossref PubMed Scopus (59) Google Scholar). Although the single most common nucleotide modification is pseudouridine, by far the most abundant type of RNA chemical modification is methylation (4Rozenski J. Crain P.F. McCloskey J.A. Nucleic Acids Res. 1999; 27: 196-197Crossref PubMed Scopus (348) Google Scholar). A vast array of unique mono-, di-, and trimethylations of each RNA base and/or ribose sugar 2′-OH is possible, and important new functions for these modifications continue to emerge. In ribosomal RNA (rRNA), 2The abbreviations used are: rRNAribosomal RNATsrthiostrepton resistance methyltransferaseAdoMetS-adenosyl-l-methionineMTasemethyltransferaseNTDN-terminal domainCTDC-terminal domainA106723 S rRNA nucleotide 1067 (E. coli numbering)SPOUTSpoU/TrmD methyltransferase familyMOPS4-morpholinepropanesulfonic acid. 2The abbreviations used are: rRNAribosomal RNATsrthiostrepton resistance methyltransferaseAdoMetS-adenosyl-l-methionineMTasemethyltransferaseNTDN-terminal domainCTDC-terminal domainA106723 S rRNA nucleotide 1067 (E. coli numbering)SPOUTSpoU/TrmD methyltransferase familyMOPS4-morpholinepropanesulfonic acid. for example, modifications cluster in functionally critical regions where methylation may act as a checkpoint in ribosome subunit assembly (5Xu Z. O'Farrell H.C. Rife J.P. Culver G.M. Nat. Struct. Mol. Biol. 2008; 15: 534-536Crossref PubMed Scopus (94) Google Scholar), influence the process of translation (6Decatur W.A. Fournier M.J. Trends Biochem. Sci. 2002; 27: 344-351Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar), and alter resistance to certain antibiotics (7Conn G.L. Savic M. Macmaster R. Grosjean H. DNA and RNA Modification Enzymes: Comparative Structure, Mechanism, Functions, Cellular Interactions and Evolution. Landes Bioscience, Austin, TX2009Google Scholar, 8Long K.S. Vester B. Grosjean H. DNA and RNA Modification Enzymes: Comparative Structure, Mechanism, Functions, Cellular Interactions and Evolution. Landes Bioscience, Austin, TX2009Google Scholar).RNA methylation is catalyzed by members of two classes (I and IV) of S-adenosyl-l-methionine (AdoMet)-dependent RNA methyltransferase (MTase) enzymes (9Schubert H.L. Blumenthal R.M. Cheng X.D. Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). In bacteria, rRNA methylations are incorporated by both “housekeeping” MTases and those that confer resistance to antibiotics. Although members of the former group are often highly conserved, the latter are generally only found in the antibiotic-producing strain as one mechanism of defense against self-intoxication (10Cundliffe E. Annu. Rev. Microbiol. 1989; 43: 207-233Crossref PubMed Scopus (335) Google Scholar). However, several instances of antibiotic resistance MTase genes in non-producer strains, including pathogenic bacteria, have been identified, and it is clear that these genes are mobile resistance determinants, usually obtained by lateral gene transfer.Several classes of antibiotics target the conserved centers on the ribosome, altering or blocking critical steps in translation such as decoding and peptidyl transfer, to exert their bactericidal effect (11Poehlsgaard J. Douthwaite S. Nat. Rev. Microbiol. 2005; 3: 870-881Crossref PubMed Scopus (402) Google Scholar). RNA MTases have been identified as clinically significant resistance determinants to a number of these, including the aminoglycoside (Arm MTase) and erythromycin (Erm MTase) antibiotics (12Doi Y. Arakawa Y. Clin. Infect. Dis. 2007; 45: 88-94Crossref PubMed Scopus (388) Google Scholar, 13Gaynor M. Mankin A.S. Curr. Top. Med. Chem. 2003; 3: 949-961Crossref PubMed Scopus (150) Google Scholar). Another functionally critical ribosome domain, the factor binding site (or “GTPase” center), is also the target for a family of thiazole-containing peptide antibiotics (14Harms J.M. Wilson D.N. Schluenzen F. Connell S.R. Stachelhaus T. Zaborowska Z. Spahn C.M. Fucini P. Mol. Cell. 2008; 30: 26-38Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), which includes thiostrepton. These antibiotics have been important biochemical tools for studies of ribosome function but are of limited clinical use due to their poor aqueous solubility. Thiostrepton is, however, used in veterinary medicine, and recent studies suggest it may have application in development of novel antimalarial and anticancer strategies (15Goodman C.D. Su V. McFadden G.I. Mol. Biochem. Parasitol. 2007; 152: 181-191Crossref PubMed Scopus (202) Google Scholar, 16Liao Z. Thibaut L. Jobson A. Pommier Y. Mol. Pharmacol. 2006; 70: 366-372Crossref PubMed Scopus (50) Google Scholar). The minimal rRNA sequence for interaction of thiostrepton is a highly conserved, independently folded 58-nucleotide rRNA domain that is also bound by ribosomal protein L11. Resistance to thiostrepton can result from mutations in the N-terminal domain of L11 or its entire absence, whereas mutation of the target nucleoside (A1067) confers far greater resistance (17Wienen B. Ehrlich R. Stöffler-Meilicke M. Stöffler G. Smith I. Weiss D. Vince R. Pestka S. J. Biol. Chem. 1979; 254: 8031-8041Abstract Full Text PDF PubMed Google Scholar, 18Bechthold A. Floss H.G. Eur. J. Biochem. 1994; 224: 431-437Crossref PubMed Scopus (34) Google Scholar, 19Cameron D.M. Thompson J. Gregory S.T. March P.E. Dahlberg A.E. Nucleic Acids Res. 2004; 32: 3220-3227Crossref PubMed Scopus (45) Google Scholar). In the thiostrepton producer Streptomyces azureus the thiostrepton resistance MTase (Tsr) catalyzes the 2′-O-methylation of A1067 resulting in specific and total resistance to thiostrepton (20Thompson J. Schmidt F. Cundliffe E. J. Biol. Chem. 1982; 257: 7915-7917Abstract Full Text PDF PubMed Google Scholar).Here we present the crystal structure of Tsr in complex with AdoMet. The structure definitively places Tsr into the SpoU/TrmD (SPOUT) family of enzymes and provides the basis for modeling the Tsr·rRNA recognition process.CONCLUSIONThe results of molecular docking experiments based on our Tsr-AdoMet complex crystal structure provide a structural rationalization for the findings of our in vitro binding and methylation assays with RNA and mutant Tsr protein. Tsr directly binds a single hairpin loop structure within the ribosomal L11-binding domain but uses each of its L30-like NTDs to recognize two distinct components of its structure: the A1067 target loop and a more distant internal bulge. Undoubtedly Tsr employs a distinct set of recognition strategies compared with L11 (32Dunstan M.S. Guhathakurta D. Draper D.E. Conn G.L. Chem. Biol. 2005; 12: 201-206Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) to bind the same rRNA domain. Like L11, however, Tsr almost certainly exploits the unique conformations in this rRNA domain to achieve absolute specificity of target selection. With the major contemporary clinical challenge of combating resistant bacterial strains, a deeper molecular understanding of the specific recognition mechanisms of rRNA resistance methyltransferases will be an essential platform for producing new designer antibiotics. RNA modifications and the enzymes that catalyze their formation are critical for cellular viability. Certain RNA modifications are extremely well characterized, such as CCA addition and amino acylation of the 3′-ends of tRNA, and the contributions of some nucleotide modifications to the creation of specific functional tRNA structures (1Gustilo E.M. Vendeix F.A. Agris P.F. Curr. Opin. Microbiol. 2008; 11: 134-140Crossref PubMed Scopus (180) Google Scholar, 2Clouet-d'Orval B. Gaspin C. Mougin A. Biochimie (Paris). 2005; 87: 889-895Crossref PubMed Scopus (33) Google Scholar, 3Ferré-D'Amaré A.R. Curr. Opin. Struct. Biol. 2003; 13: 49-55Crossref PubMed Scopus (59) Google Scholar). Although the single most common nucleotide modification is pseudouridine, by far the most abundant type of RNA chemical modification is methylation (4Rozenski J. Crain P.F. McCloskey J.A. Nucleic Acids Res. 1999; 27: 196-197Crossref PubMed Scopus (348) Google Scholar). A vast array of unique mono-, di-, and trimethylations of each RNA base and/or ribose sugar 2′-OH is possible, and important new functions for these modifications continue to emerge. In ribosomal RNA (rRNA), 2The abbreviations used are: rRNAribosomal RNATsrthiostrepton resistance methyltransferaseAdoMetS-adenosyl-l-methionineMTasemethyltransferaseNTDN-terminal domainCTDC-terminal domainA106723 S rRNA nucleotide 1067 (E. coli numbering)SPOUTSpoU/TrmD methyltransferase familyMOPS4-morpholinepropanesulfonic acid. 2The abbreviations used are: rRNAribosomal RNATsrthiostrepton resistance methyltransferaseAdoMetS-adenosyl-l-methionineMTasemethyltransferaseNTDN-terminal domainCTDC-terminal domainA106723 S rRNA nucleotide 1067 (E. coli numbering)SPOUTSpoU/TrmD methyltransferase familyMOPS4-morpholinepropanesulfonic acid. for example, modifications cluster in functionally critical regions where methylation may act as a checkpoint in ribosome subunit assembly (5Xu Z. O'Farrell H.C. Rife J.P. Culver G.M. Nat. Struct. Mol. Biol. 2008; 15: 534-536Crossref PubMed Scopus (94) Google Scholar), influence the process of translation (6Decatur W.A. Fournier M.J. Trends Biochem. Sci. 2002; 27: 344-351Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar), and alter resistance to certain antibiotics (7Conn G.L. Savic M. Macmaster R. Grosjean H. DNA and RNA Modification Enzymes: Comparative Structure, Mechanism, Functions, Cellular Interactions and Evolution. Landes Bioscience, Austin, TX2009Google Scholar, 8Long K.S. Vester B. Grosjean H. DNA and RNA Modification Enzymes: Comparative Structure, Mechanism, Functions, Cellular Interactions and Evolution. Landes Bioscience, Austin, TX2009Google Scholar). ribosomal RNA thiostrepton resistance methyltransferase S-adenosyl-l-methionine methyltransferase N-terminal domain C-terminal domain 23 S rRNA nucleotide 1067 (E. coli numbering) SpoU/TrmD methyltransferase family 4-morpholinepropanesulfonic acid. ribosomal RNA thiostrepton resistance methyltransferase S-adenosyl-l-methionine methyltransferase N-terminal domain C-terminal domain 23 S rRNA nucleotide 1067 (E. coli numbering) SpoU/TrmD methyltransferase family 4-morpholinepropanesulfonic acid. RNA methylation is catalyzed by members of two classes (I and IV) of S-adenosyl-l-methionine (AdoMet)-dependent RNA methyltransferase (MTase) enzymes (9Schubert H.L. Blumenthal R.M. Cheng X.D. Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). In bacteria, rRNA methylations are incorporated by both “housekeeping” MTases and those that confer resistance to antibiotics. Although members of the former group are often highly conserved, the latter are generally only found in the antibiotic-producing strain as one mechanism of defense against self-intoxication (10Cundliffe E. Annu. Rev. Microbiol. 1989; 43: 207-233Crossref PubMed Scopus (335) Google Scholar). However, several instances of antibiotic resistance MTase genes in non-producer strains, including pathogenic bacteria, have been identified, and it is clear that these genes are mobile resistance determinants, usually obtained by lateral gene transfer. Several classes of antibiotics target the conserved centers on the ribosome, altering or blocking critical steps in translation such as decoding and peptidyl transfer, to exert their bactericidal effect (11Poehlsgaard J. Douthwaite S. Nat. Rev. Microbiol. 2005; 3: 870-881Crossref PubMed Scopus (402) Google Scholar). RNA MTases have been identified as clinically significant resistance determinants to a number of these, including the aminoglycoside (Arm MTase) and erythromycin (Erm MTase) antibiotics (12Doi Y. Arakawa Y. Clin. Infect. Dis. 2007; 45: 88-94Crossref PubMed Scopus (388) Google Scholar, 13Gaynor M. Mankin A.S. Curr. Top. Med. Chem. 2003; 3: 949-961Crossref PubMed Scopus (150) Google Scholar). Another functionally critical ribosome domain, the factor binding site (or “GTPase” center), is also the target for a family of thiazole-containing peptide antibiotics (14Harms J.M. Wilson D.N. Schluenzen F. Connell S.R. Stachelhaus T. Zaborowska Z. Spahn C.M. Fucini P. Mol. Cell. 2008; 30: 26-38Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), which includes thiostrepton. These antibiotics have been important biochemical tools for studies of ribosome function but are of limited clinical use due to their poor aqueous solubility. Thiostrepton is, however, used in veterinary medicine, and recent studies suggest it may have application in development of novel antimalarial and anticancer strategies (15Goodman C.D. Su V. McFadden G.I. Mol. Biochem. Parasitol. 2007; 152: 181-191Crossref PubMed Scopus (202) Google Scholar, 16Liao Z. Thibaut L. Jobson A. Pommier Y. Mol. Pharmacol. 2006; 70: 366-372Crossref PubMed Scopus (50) Google Scholar). The minimal rRNA sequence for interaction of thiostrepton is a highly conserved, independently folded 58-nucleotide rRNA domain that is also bound by ribosomal protein L11. Resistance to thiostrepton can result from mutations in the N-terminal domain of L11 or its entire absence, whereas mutation of the target nucleoside (A1067) confers far greater resistance (17Wienen B. Ehrlich R. Stöffler-Meilicke M. Stöffler G. Smith I. Weiss D. Vince R. Pestka S. J. Biol. Chem. 1979; 254: 8031-8041Abstract Full Text PDF PubMed Google Scholar, 18Bechthold A. Floss H.G. Eur. J. Biochem. 1994; 224: 431-437Crossref PubMed Scopus (34) Google Scholar, 19Cameron D.M. Thompson J. Gregory S.T. March P.E. Dahlberg A.E. Nucleic Acids Res. 2004; 32: 3220-3227Crossref PubMed Scopus (45) Google Scholar). In the thiostrepton producer Streptomyces azureus the thiostrepton resistance MTase (Tsr) catalyzes the 2′-O-methylation of A1067 resulting in specific and total resistance to thiostrepton (20Thompson J. Schmidt F. Cundliffe E. J. Biol. Chem. 1982; 257: 7915-7917Abstract Full Text PDF PubMed Google Scholar). Here we present the crystal structure of Tsr in complex with AdoMet. The structure definitively places Tsr into the SpoU/TrmD (SPOUT) family of enzymes and provides the basis for modeling the Tsr·rRNA recognition process. CONCLUSIONThe results of molecular docking experiments based on our Tsr-AdoMet complex crystal structure provide a structural rationalization for the findings of our in vitro binding and methylation assays with RNA and mutant Tsr protein. Tsr directly binds a single hairpin loop structure within the ribosomal L11-binding domain but uses each of its L30-like NTDs to recognize two distinct components of its structure: the A1067 target loop and a more distant internal bulge. Undoubtedly Tsr employs a distinct set of recognition strategies compared with L11 (32Dunstan M.S. Guhathakurta D. Draper D.E. Conn G.L. Chem. Biol. 2005; 12: 201-206Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) to bind the same rRNA domain. Like L11, however, Tsr almost certainly exploits the unique conformations in this rRNA domain to achieve absolute specificity of target selection. With the major contemporary clinical challenge of combating resistant bacterial strains, a deeper molecular understanding of the specific recognition mechanisms of rRNA resistance methyltransferases will be an essential platform for producing new designer antibiotics. The results of molecular docking experiments based on our Tsr-AdoMet complex crystal structure provide a structural rationalization for the findings of our in vitro binding and methylation assays with RNA and mutant Tsr protein. Tsr directly binds a single hairpin loop structure within the ribosomal L11-binding domain but uses each of its L30-like NTDs to recognize two distinct components of its structure: the A1067 target loop and a more distant internal bulge. Undoubtedly Tsr employs a distinct set of recognition strategies compared with L11 (32Dunstan M.S. Guhathakurta D. Draper D.E. Conn G.L. Chem. Biol. 2005; 12: 201-206Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) to bind the same rRNA domain. Like L11, however, Tsr almost certainly exploits the unique conformations in this rRNA domain to achieve absolute specificity of target selection. With the major contemporary clinical challenge of combating resistant bacterial strains, a deeper molecular understanding of the specific recognition mechanisms of rRNA resistance methyltransferases will be an essential platform for producing new designer antibiotics.

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