title: A Novel Motif for S-Adenosyl-l-methionine Binding by the Ribosomal RNA Methyltransferase TlyA from Mycobacterium tuberculosis.
abstract: Capreomycin is a potent ribosome-targeting antibiotic that is an essential component of current antituberculosis treatments, particularly in the case of multidrug-resistant Mycobacterium tuberculosis (Mtb). Optimal capreomycin binding and Mtb ribosome inhibition requires ribosomal RNA methylation in both ribosome subunits by TlyA (Rv1694), an enzyme with dual 2'-O-methytransferase and putative hemolytic activities. Despite the important role of TlyA in capreomycin sensitivity and identification of inactivating mutations in the corresponding Mtb gene tlyA, which cause resistance to capreomycin, our current structural and mechanistic understanding of TlyA action remains limited. Here, we present structural and functional analyses of Mtb TlyA interaction with its obligatory co-substrate for methyltransferase activity, S-adenosyl-l-methionine (SAM). Despite adopting a complete class I methyltransferase fold containing conserved SAM-binding and catalytic motifs, the isolated TlyA carboxyl-terminal domain exhibits no detectable affinity for SAM. Further analyses identify a tetrapeptide motif (RXWV) in the TlyA interdomain linker as indispensable for co-substrate binding. Our results also suggest that structural plasticity of the RXWV motif could contribute to TlyA domain interactions, as well as specific recognition of its two structurally distinct ribosomal RNA targets. Our findings thus reveal a novel motif requirement for SAM binding by TlyA and set the stage for future mechanistic studies of TlyA substrate recognition and modification that underpin Mtb sensitivity to capreomycin.
authors.affiliation: From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: Marta A
authors.initials: MA
authors.lastname: Witek
authors.affiliation: From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322; Department of Biochemistry, Cell and Developmental Biology Program, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: Emily G
authors.initials: EG
authors.lastname: Kuiper
authors.affiliation: Department of Biochemistry, Cell and Developmental Biology Program, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Minten
authors.affiliation: the Microbiology and Molecular Genetics Program, Graduate Division of Biological and Biomedical Sciences, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: Emily K
authors.initials: EK
authors.lastname: Crispell
authors.affiliation: From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322. Electronic address: gconn@emory.edu.
authors.forename: Graeme L
authors.initials: GL
authors.lastname: Conn
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002207:Capreomycin / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C112817:TlyA protein, Mycobacterium tuberculosis; D002207:Capreomycin; D012436:S-Adenosylmethionine
keywords: Mycobacterium tuberculosis; RNA methylation; RNA methyltransferase; S-adenosylmethionine (SAM); X-ray crystallography; antibiotic resistance; isothermal titration calorimetry (ITC); protein motif; proteolysis; site-directed mutagenesis