RESUMEN
DNA methylation, an important epigenetic modification, is catalyzed by DNA methyltransferases and is essential in the regulation of gene expression. Here, the utility of an N-mustard analog designed to mimic the native methyl donor, S-adenosyl-L-methionine (SAM), was explored with the DNA methyltransferase 3A catalytic domain (DNMT3AC). In lieu of the expected analog transfer to DNA, methyltransferase activity was instead inhibited in a concentration dependent manner. Further investigation into the mechanism of analog inhibition did not reveal a typical competitive mechanism. Instead, mass spectrometry analysis provided direct evidence of two cysteine residues in the SAM binding site covalently modified by the SAM analog and confirmed its' function as an irreversible inhibitor of DNMT3AC.
RESUMEN
The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.
Asunto(s)
Conformación de Ácido Nucleico , Nucleótidos , ARN de Transferencia , Saccharomyces cerevisiae , ARNt Metiltransferasas , Humanos , Nucleótidos/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , ARNt Metiltransferasas/química , ARNt Metiltransferasas/metabolismoRESUMEN
Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine analog to trap the complex in a postcatalytic state to enable determination of a global 3.0 Å cryo-electron microscopy structure of the m7G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder-to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m7G1405 modification to resensitize bacterial pathogens to aminoglycosides.
Asunto(s)
Aminoglicósidos , Antibacterianos , ARN Ribosómico 16S , Microscopía por Crioelectrón , Metiltransferasas , ARN Ribosómico , Escherichia coliRESUMEN
Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m 7 G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine (SAM) analog to trap the complex in a post-catalytic state to enable determination of an overall 3.0 Ã cryo-electron microscopy structure of the m 7 G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m 7 G1405 modification to re-sensitize bacterial pathogens to aminoglycosides.
RESUMEN
The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the 9th nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 14 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) which allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.
RESUMEN
Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.
Asunto(s)
Proteínas Bacterianas , Metiltransferasas , Mycobacterium tuberculosis , Subunidades Ribosómicas Grandes Bacterianas , Proteínas Bacterianas/química , Dominio Catalítico , Farmacorresistencia Bacteriana/genética , Metiltransferasas/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Conformación Proteica en Hélice alfa , ARN Ribosómico 16S/química , ARN Ribosómico 23S/química , Subunidades Ribosómicas Grandes Bacterianas/químicaRESUMEN
Histones, the fundamental building blocks of nucleosomes, undergo post-translational modifications and play a major role in the regulation of transcriptional processes. Although the significance of these modifications, including methylation, is widely recognized, little is known about the mechanisms connecting such events. To improve our understanding of how protein methylation is intricately linked, we have developed novel N-mustard analogues of S-adenosyl-l-methionine (SAM) functionalized with azides and alkynes to serve as probes of biological methylation. Here, we demonstrate their ability to serve as effective cofactor mimics of SAM and to be enzymatically transferred by protein arginine methyltransferaseâ 1 (PRMT1) to histone H4 with high site selectively for its target Arg3 on the histone tail. Further incorporation of biotin through copper-catalyzed click chemistry permitted visualization and isolation of the analogue-modified histone H4 from a complex mixture. This work validates the future utility of N-mustard analogues as probes of protein methylation events beyond PRMT1.
Asunto(s)
Histonas/aislamiento & purificación , Planta de la Mostaza/química , Proteína-Arginina N-Metiltransferasas/química , Proteínas Represoras/química , S-Adenosilmetionina/química , Química Clic , Histonas/química , Histonas/metabolismo , Humanos , Planta de la Mostaza/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Nucleosomes, the fundamental building blocks of eukaryotic chromatin, undergo post-synthetic modifications and play a major role in the regulation of transcriptional processes. Combinations of these modifications, including methylation, regulate chromatin structure, determining its different functional states and playing a central role in differentiation. The biological significance of cellular methylation, particularly on chromatin, is widely recognized, yet we know little about the mechanisms that link biological methylation events. To characterize and fully understand protein methylation, we describe here novel N-mustard analogs of S-adenosyl-l-methionine (SAM) as biochemical tools to better understand protein arginine methylation events using protein arginine methyltransferase 1 (PRMT1). Specifically, azide- and alkyne-functionalized N-mustard analogs serve as cofactor mimics of SAM and are enzymatically transferred to a model peptide substrate in a PRMT1-dependent fashion. Once incorporated, the resulting alkynes and azides can be modified through chemoselective ligations, including click chemistry and the Staudinger ligation. These results readily demonstrate the feasibility of utilizing N-mustard analogs as biochemical tools to site-specifically label substrates of PRMT1 and serve as an alternative approach to study protein methylation events.
Asunto(s)
Arginina/metabolismo , S-Adenosilmetionina/análogos & derivados , Alquinos/química , Secuencia de Aminoácidos , Arginina/química , Azidas/química , Biotinilación , Cromatografía Líquida de Alta Presión , Química Clic , Humanos , Metilación , Datos de Secuencia Molecular , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , S-Adenosilmetionina/análisis , S-Adenosilmetionina/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Azide and alkyne-functionalized N-mustard analogues of S-adenosyl-L-methionine have been synthesized and were demonstrated to undergo efficient methyltransferase-dependent DNA alkylation by M.TaqI and M.HhaI. Subsequent labeling of the DNA with a fluorophore was carried out using copper-catalyzed azide-alkyne cycloaddition chemistry and was visualized by fluorescence scanning. This work demonstrates the utility of functionalized N-mustard analogues as biochemical tools to study biological methylation and offers a facile way to site-selectively label substrates of DNA methyltransferases.
Asunto(s)
Alquinos/química , Azidas/química , ADN/análisis , Colorantes Fluorescentes/análisis , S-Adenosilmetionina/análogos & derivados , Alquilación , Alquinos/metabolismo , Azidas/metabolismo , Química Clic , ADN/metabolismo , Colorantes Fluorescentes/metabolismo , Plásmidos/análisis , Plásmidos/metabolismo , S-Adenosilmetionina/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismoRESUMEN
The synthesis of an azide-bearing N-mustard S-adenosyl-L-methionine (SAM) analogue, 8-azido-5'-(diaminobutyric acid)-N-iodoethyl-5'-deoxyadenosine, has been accomplished in 10 steps from commercially available 2',3'-isopropylidene adenosine. Critical to this success was executing C8 azidation prior to derivatizing the 5'-position of the ribose sugar and the late stage alkylation of the 5' amino group with bromoethanol, which was necessitated by the reactivity of the aryl azide moiety. The azide-bearing N-mustard is envisioned as a useful biochemical tool by which to probe DNA and protein methylation patterns.
Asunto(s)
Azidas/química , Sondas Moleculares/síntesis química , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/síntesis química , Alquilación , Técnicas de Química Sintética , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Desoxiadenosinas/química , Humanos , Metilación , Compuestos de Mostaza/química , Ribosa/químicaRESUMEN
O-acetyl-ADP-ribose (OAADPr), produced by the Sir2-catalyzed NAD(+)-dependent histone/protein deacetylase reaction, regulates diverse biological processes. Interconversion between two OAADPr isomers with acetyl attached to the C-2â³ and C-3â³ hydroxyl of ADP-ribose (ADPr) is rapid. We reported earlier that ADP-ribosylhydrolase 3 (ARH3), one of three ARH proteins sharing structural similarities, hydrolyzed OAADPr to ADPr and acetate, and poly(ADPr) to ADPr monomers. ARH1 also hydrolyzed OAADPr and poly(ADPr) as well as ADP-ribose-arginine, with arginine in α-anomeric linkage to C-1â³ of ADP-ribose. Because both ARH3- and ARH1-catalyzed reactions involve nucleophilic attacks at the C-1â³ position, it was perplexing that the ARH3 catalytic site would cleave OAADPr at either the 2â³- or 3â³-position, and we postulated the existence of a third isomer, 1â³-OAADPr, in equilibrium with 2â³- and 3â³-isomers. A third isomer, consistent with 1â³-OAADPr, was identified at pH 9.0. Further, ARH3 OAADPr hydrolase activity was greater at pH 9.0 than at neutral pH where 3â³-OAADPr predominated. Consistent with our hypothesis, IC(50) values for ARH3 inhibition by 2â³- and 3â³-N-acetyl-ADPr analogs of OAADPr were significantly higher than that for ADPr. ARH1 also hydrolyzed OAADPr more rapidly at alkaline pH, but cleavage of ADP-ribose-arginine was faster at neutral pH than pH 9.0. ARH3-catalyzed hydrolysis of OAADPr in H(2)(18)O resulted in incorporation of one (18)O into ADP-ribose by mass spectrometric analysis, consistent with cleavage at the C-1â³ position. Together, these data suggest that ARH family members, ARH1 and ARH3, catalyze hydrolysis of the 1â³-O linkage in their structurally diverse substrates.
Asunto(s)
Glicósido Hidrolasas/química , N-Glicosil Hidrolasas/química , O-Acetil-ADP-Ribosa/metabolismo , Adenosina Difosfato Ribosa/química , Catálisis , Dominio Catalítico , Regulación Enzimológica de la Expresión Génica , Concentración de Iones de Hidrógeno , Hidrólisis , Concentración 50 Inhibidora , Modelos Químicos , Modelos Teóricos , Poli Adenosina Difosfato Ribosa/química , Isoformas de Proteínas , Sirtuina 1/química , Sirtuinas/químicaRESUMEN
Synthetic routes for the preparation of O-acetyl-ADP-ribose and two novel non-hydrolyzable analogs containing an N-acetyl are described and shown to interact with the macro domain of histone protein H2A1.1.
Asunto(s)
Adenosina Difosfato Ribosa/biosíntesis , O-Acetil-ADP-Ribosa/biosíntesis , Acetilación , Modelos Moleculares , O-Acetil-ADP-Ribosa/química , Sirtuinas/metabolismoRESUMEN
We demonstrate here that MTase-modified DNA can undergo the Staudinger ligation with triarylphosphines derivatized with phenanthroline. Presentation of these duplexes with Cu(II) and 3-mercaptopropionic acid leads to strand scission proximal to the MTase recognition site. By virtue of their ability to use a synthetic azide-bearing cofactor, M.TaqI and M.HhaI produce a DNA lesion that induces scission 5' to the base modified by the enzyme. This chemistry represents a new approach by which regions of DNA methylation can be rapidly identified on the basis of DNA damage.
Asunto(s)
ADN/química , Metiltransferasas/química , Animales , Daño del ADN , ADN de Cadena Simple , Metilación , Estructura MolecularRESUMEN
Aziridine-based cofactor mimics have been synthesized and are shown to undergo methyltransferase-dependent DNA alkylation. Notably, each cofactor mimic possesses an azide functionality, to which can be attached an assortment of unnatural groups following methyltransferase-dependent DNA delivery. DNA duplexes modified with these cofactor mimics are capable of undergoing the Staudinger ligation with phosphines tethered to biological functionalities following enzymatic modification. This methodology provides a new tool by which to selectively modify DNA in a methyltransferase-dependent way. The conversion of biological methyltransferases into azidonucleosidyl transferases demonstrated here also holds tremendous promise as a means of identifying, as yet, unknown substrates of methylation.
Asunto(s)
Azidas/química , Aziridinas/química , Coenzimas/química , ADN/metabolismo , Nucleósidos/metabolismo , Pentosiltransferasa/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Alquilación , Biotinilación , ADN/química , Imitación Molecular , Nucleósidos/síntesis química , Nucleósidos/química , Oligonucleótidos/química , Oligonucleótidos/metabolismoRESUMEN
8-Azido-5'-aziridino-5'-deoxyadenosine (6), a novel cofactor mimic, was synthesized in nine steps from commercially available 2',3'-isopropylideneadenosine in approximately 4% overall yield. Crucial to this success was a very unorthodox phthalimide cleavage procedure, C8 azidation prior to aziridination and late stage alkylation of the 5' amino group with iodoethanol necessitated by the high degree of lability endowed by the aryl azide moiety. Aziridine 6 is envisioned as a useful biochemical tool by which to probe DNA and protein methylation patterns.
Asunto(s)
Adenosina/análogos & derivados , Adenosina/química , Adenosina/síntesis química , Azidas/síntesis química , Aziridinas/síntesis química , Sondas Moleculares/síntesis química , Azidas/química , Aziridinas/química , Sondas Moleculares/químicaRESUMEN
[reaction: see text] o-Carboalkoxy triarylphosphines are shown to react with aryl azides to provide Staudinger ligation products bearing O-alkyl imidate linkages. This is in contrast to alkyl azides whose ligation to o-carboalkoxy triarylphosphines has been reported to yield amide-linked materials. This extension of the Staudinger ligation for coupling of abiotic reagents under biocompatible conditions highlights the utility of commercially available triarylphosphines through which suitable linkers can be attached via an ester moiety.