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.

Biofilm Formation and Motility Are Promoted by Cj0588-Directed Methylation of rRNA in Campylobacter jejuni

“…Sequences were aligned with Clustal Omega (Sievers et al, 2011 ). (B) Model of the catalytic domain of the Cj0588 structure based on the 1.7 Å resolution crystal structure of the M. tuberculosis TlyA (PDB: 5EOV) (Witek et al, 2017 ). The N-terminal S4 RNA binding domain is missing in the structure (Witek et al, 2017 ).…”

“…(B) Model of the catalytic domain of the Cj0588 structure based on the 1.7 Å resolution crystal structure of the M. tuberculosis TlyA (PDB: 5EOV) (Witek et al, 2017 ). The N-terminal S4 RNA binding domain is missing in the structure (Witek et al, 2017 ). The catalytic tetrad (blue); in this study, three of these residues, K80, D162 and K188, were individually substituted with alanine.…”

“…Sequences were aligned with Clustal Omega (Sievers et al, 2011 ). Modeling of the tertiary structure of the Cj0588 catalytic domain was carried out with Modeller version 9.19 (Sali and Blundell, 1993 ) in UCSF Chimera (Pettersen et al, 2004 ) using M. tuberculosis TlyA (PDB: 5EOV) (Witek et al, 2017 ) as a reference to fold the Cj0588 peptide chain. The HhaI methyltransferase structure (PDB: 2HMY) was used to superimpose the AdoMet cofactor (O'Gara et al, 1999 ).…”

Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition

“…Equivalent expression constructs for RmtD (Uniprot ID: B0F9V0) and RmtD2 (Uniprot ID: A0A0U3JA93) were previously reported (35). Variants of RmtC were prepared using the megaprimer whole-plasmid PCR method (13,36) and confirmed by automated DNA sequencing. Expression of all wild-type methyltransferases and variant RmtC proteins from the modified pET44 vector produced proteins with an N-terminal 6xHis tag and thrombin protease recognition sequence.…”

The Evolution of Substrate Specificity by tRNA Modification Enzymes

“…The other known conserved SAM-binding residue is an acidic residue at the end of β2, which forms hydrogen bonds with both hydroxyls of the SAM ribose [30]. Recently, in the TlyA ribosomal RNA methyltransferase from Mycobacterium tuberculosis an additional novel SAM-binding motif, RxWV, was discovered [37], which affects the structure of a GxGxG motif in a purposeful way [37], and both motifs are required for SAM binding [37]. Still, in general the SAM-binding region normally resides within the N-terminus of many methyltransferases and is assembled primarily from loops following strands 1, 2, and 3, whereas the substrate-binding region is generally located in the C-terminal portion of the β-sheet [29,38].…”