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. 2017 Feb 3;292(5):1977-1987.
doi: 10.1074/jbc.M116.752659. Epub 2016 Dec 27.

A Novel Motif for S-Adenosyl-l-methionine Binding by the Ribosomal RNA Methyltransferase TlyA from Mycobacterium tuberculosis

Marta A Witek  1 , Emily G Kuiper  2 , Elizabeth Minten  3 , Emily K Crispell  4 , Graeme L Conn  5
Affiliations

A Novel Motif for S-Adenosyl-l-methionine Binding by the Ribosomal RNA Methyltransferase TlyA from Mycobacterium tuberculosis

Marta A Witek et al. J Biol Chem. .

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.

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.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
Recombinant Mtb His-TlyA (Rv1694) is well folded and active in vitro against both 30S and 50S ribosomal subunit substrates. A, CD spectrum of His-TlyA and results of secondary structural deconvolution (inset). MRW, mean residue molar ellipticity. B, RT analysis of in vitro 2′-O-methylation by TlyA (solid arrowheads) of residues C1409 (16S rRNA, left) and C1920 (23S rRNA, right). The positions of nearby modifications normally present in E. coli are also indicated (open arrowheads).
FIGURE 2.
FIGURE 2.
GluC cleavage of TlyA generates two domain fragments that remain associated. A, SDS-PAGE of His-TlyA (black arrow) and stable fragments generated by GluC cleavage: CTDGluC (gray arrow) and NTDGluC (blue arrow); M is protein gel standard (masses in kDa are shown to the left). B, homology model (PM0076044) of TlyA highlighting the locations of the proposed GluC cleavage site (Glu59; red), the tetrapeptide interdomain linker (RAWV motif; orange), and the predicted SAM (yellow) binding site modeled using the structure of HhaI methyltransferase (PDB code 2HMY). C, time course of a partial digest of WT and E59A substituted TlyA with GluC protease. Stable TlyA fragments generated after GluC cleavage and molecular mass standards are indicated as in A. D, gel filtration chromatogram of His-TlyA (black) and TlyA partially digested with GluC (gray). E, SDS-PAGE of pooled fractions from peak 1 indicating CTDGluC and NTDGluC as in A. F, schematic diagrams of full-length TlyA and individual domain expression constructs highlighting the proposed GluC cleavage (red arrow), thrombin cleavable hexahistidine tag (black arrow), and amino acids retained after thrombin cleavage (lowercase text), and the Ulp cleavable SUMO domain (white arrow). Solid and dashed lines for the amino-terminal tags (His and SUMO) denote those retained and cleaved in the expressed proteins used in the present studies, respectively.
FIGURE 3.
FIGURE 3.
The TlyA CTD adopts a class I methyltransferase fold. A, two orthogonal views of the TlyA CTD crystal structure with the conserved seven-stranded β-sheet core (left) and six surrounding α-helices (right) highlighted in purple. B, model of the TlyA CTD interaction with SAM (yellow; from PDB 2HMY) shown in the same orientation as A. Residues highlighted are from the SAM binding motif I 90GASTG94 (pink backbone, purple side chains) and Asp154 of the proposed TlyA catalytic tetrad (green). C, amino acids proposed to make up the SAM binding pocket in TlyA, colored as in B.
FIGURE 4.
FIGURE 4.
The isolated TlyA methyltransferase domain (CTD) does not bind SAM. ITC analysis of TlyA-SAM interaction for titration by SAM into the cell containing His-TlyA (A), tag-free CTD (B), and His-TlyA after cleavage with GluC (His-TlyAGluC) (C).
FIGURE 5.
FIGURE 5.
The TlyA RAWV tetrapeptide sequence is necessary for SAM binding. A, gel filtration analysis of His-TlyA (black) and individually expressed and purified, tag-free NTDRAWV and CTD (gray dashed line). Inset, SDS-PAGE of pooled fractions from peaks 1 and 2. B, SDS-PAGE of sample enriched for TlyA CTD derived from GluC cleavage of full-length His-TlyA (CTDGluC). C, ITC analysis of SAM binding to the CTDGluC fragment. D, ITC analysis of SAM binding to the RAWVCTD protein construct. E, gel filtration chromatogram of His-TlyA (black) and individually expressed and purified, tag-free NTD and RAWVCTD (gray dashed line). Inset, SDS-PAGE of pooled fractions from peaks 1 and 2.
FIGURE 6.
FIGURE 6.
RXWV motif conservation. A, WebLogo representation of RAWV conservation of the top 250 homolog sequences retrieved using a BLAST search with the TlyA-RAWVCTD sequence in UniProt. Additional WebLogo representations for subsets of these 250 homologs. B, all SAM-dependent (methyltransferase) proteins. C, TlyA family members from Mycobacteria (sequence identity 68–100%). D, TlyA family members from species other than Mycobacteria (sequence identity 66–68%). E, conservation of the RAWV sequences among TlyA homologs functionally characterized by Monshupanee et al. (identity 38–100%) (25, 37).
FIGURE 7.
FIGURE 7.
The TlyA RAWV motif can adopt two distinct conformations. A, cartoon of form 1 loop RAWVCTD structure. The amino acids side chains of Trp62 and Val63 are shown as orange sticks. B, zoomed view of Trp62 and Val63 in form 1 loop shown in 2mFo − DFc omit electron density contoured at 1σ. C, form 2 helix structure shown as a cartoon with all side chains of the RAWV sequence shown as sticks (cyan). D, zoomed view of the helical RAWV sequence shown in 2mFo − DFc omit electron density contoured at 1σ. E, overlay of form 1 loop with two hemolysin structures (PDB code 3HP7 and 3OPN, blue and green, respectively). F, superimposition of TlyA CTD (purple), form 1 loop (tan), and form 2 helix (teal) structures modeled with SAM (from PDB code 2HMY; yellow). Val63 and Trp62 are as colored as in A and C. Additional residues which differ in their Cα positions between the two structures are shown as spheres. G, zoomed view of Val63 and Thr92 reorientation toward the modeled SAM. H and I, zoomed views of Thr93 of form 1 loop (H) or form 2 helix (I), shown in 2mFo − DFc omit electron density contoured at 1σ.
FIGURE 8.
FIGURE 8.
Modeling of the TlyA NTD on the two RAWVCTD crystal structures. Two approximately orthogonal views of the overlaid TlyA NTD modeled onto the form 1 loop (orange NTD) and the form 2 helix (teal NTD).
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