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

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.

population and resulted in an estimated 9.6 million new cases of active TB disease and 1.5 million deaths in 2014 (1). Of further significant concern is the rising number of TB cases involving Mtb strains that are either multidrug-resistant, defined as being resistant to the first line antibiotics isoniazid and rifampicin, or extensively drug-resistant, defined as being additionally resistant to any fluoroquinolone and at least one of the three injectable second line drugs: amikacin, kanamycin, or capreomycin (2,3).
The cyclic aminoglycoside-like peptide antibiotic capreomycin targets the mycobacterial ribosome at the interface of the small and large subunits (4) and requires ribosomal RNA (rRNA) methylation for optimal binding and thus inhibition of ribosome function. Resistance to capreomycin in Mtb can arise via mutation of tlyA, the gene encoding the protein TlyA (Rv1694), a proposed virulence factor for Mtb with dual hemolytic and rRNA methyltransferase activities (5)(6)(7)(8). Resistance to ribosome-targeting drugs is generally associated with the addition of methyl groups rather than their loss (9 -11). Thus, TlyA belongs to a unique group of methyltransferases for which the loss of function confers bacterial antibiotic resistance. Additionally, because many bacterial genera lack tlyA, the potent antibiotic activity of capreomycin is specific against Mtb (6,12). However, treatment of TB has become problematic not only because of the side effects of aminoglycosides but also because of the increased incidence of virulent, capreomycin-resistant Mtb strains generated by inactivation of tlyA (6,13). Despite the critical role of TlyA in capreomycin sensitivity and identification of inactivating mutations that cause resistance, our current understanding of TlyA structure and mechanism of action remains limited.
The S-adenosyl-L-methionine (SAM)-dependent methyltransferase activity of TlyA results in ribose 2Ј-OH methylation of two cytidine residues: 16S rRNA C1409, which is located within the 30S (small) ribosomal subunit "decoding center," and 23S rRNA C1920, which is present in a highly conserved region of the 50S (large) ribosomal subunit near the subunit interface (4,6,14). Despite the importance of TlyA methyltransferase activity in capreomycin action and resistance, many molecular details of the TlyA mechanism of action remain largely unknown, including interaction with co-substrate SAM and how TlyA recognizes and methylates its two structurally distinct substrates (23S and 16S rRNA). Therefore, detailed molecular studies of TlyA are urgently required to better understand this resistance determinant and its contribution to capreomycin susceptibility.
Here, we demonstrate experimentally that TlyA folds into two stable structural domains connected by a protease-sensitive linker, with rRNA binding and SAM binding/methyltransferase activities expected to reside in the amino-and carboxylterminal domains (NTD and CTD), respectively. A high resolution X-ray crystal structure of the TlyA CTD reveals a Class I methyltransferase fold containing all expected conserved SAM binding and catalytic motifs. Surprisingly, however, this isolated protein domain has no detectable affinity for SAM. Further structural and functional studies reveal a novel tetrapeptide motif (RXWV) in the region linking the two structural domains as indispensable for co-substrate binding. Finally, our results also suggest that structural plasticity within this interdomain linker could play a role in TlyA recognition of its two structurally distinct rRNA targets.

Results
Construct Design, Protein Expression, and Purification of TlyA for Structural Studies-A plasmid encoding amino-terminally hexahistidine-tagged Mtb TlyA (His-TlyA) was generated for heterologous expression in Escherichia coli, and the resulting protein was purified to near homogeneity using Ni 2ϩ affinity and gel filtration chromatographies. The CD spectrum of His-TlyA is consistent with that of a well folded protein with a mixed ␣/␤ structure (Fig. 1A). We additionally showed our purified recombinant His-TlyA to be active in methylation of both target nucleotides, C1920 (23S rRNA) and C1409 (16S rRNA), by primer extension analysis of in vitro methylated E. coli 50S and 30S subunits (Fig. 1B).
We next attempted to crystallize full-length His-TlyA to solve its high resolution X-ray crystal structure, but efforts to obtain suitable crystals were unsuccessful. Therefore, His-TlyA was treated with various proteases with the goal of identifying stable fragment(s) of TlyA better suited to structural studies. The endopeptidase GluC, a serine proteinase that selectively cleaves peptide bonds carboxyl-terminal to glutamic acid residues (15), produced two stable fragments of ϳ10 and ϳ20 kDa ( Fig. 2A). Based on the observed digestion pattern and inspection of the TlyA sequence and homology model (16), we identified glutamic acid 59 (Glu 59 ) as the most likely site of GluC cleavage. The TlyA homology model predicts Glu 59 to be surface-exposed in an unstructured region that links the predicted NTD and CTD (Fig. 2B), and GluC cleavage after this residue would result in products of 7.9 or 6.2 kDa (NTD; with or without the hexhistidine tag) and 21.8 kDa (CTD), correlating well with the observed products. Cleavage products of similar sizes were also previously observed for TlyA treated with proteinase K, which was hypothesized to target a site within the interdomain linker (5). To test whether Glu 59 was indeed the GluC cleavage site, a TlyA-E59A variant was generated, and the purified protein similarly was subjected to GluC cleavage. Although GluC cleavage was not abolished, the pattern of fragments produced from the variant protein was altered, suggesting that Glu 59 is the major, but not the only, cleavage site recognized by the protease (Fig. 2C). We next asked whether these two GluC-derived TlyA fragments remain stably folded and associated by applying partially cleaved protein to a gel filtration column. Two major peaks were observed (Fig. 2D), with the earlier eluting protein corresponding to the remaining uncleaved full-length His-TlyA protein. Although eluting at a volume corresponding to a significantly lower molecular mass entity, the later peak was found to contain both stable fragments (Fig. 2, D and E), suggesting that the TlyA NTD and CTD remain stably folded and associated following GluC cleavage.
We conclude from these results that treatment of His-TlyA with GluC produces two stable protein fragments that likely correspond to the predicted TlyA NTD and CTD and that these domains have sufficient affinity that they remain associated following cleavage under the solution conditions used. Based on these observations, we generated a new expression construct for crystallographic studies of the TlyA methyltransferase domain (CTD) beginning at residue Ser 64 , which immediately follows the predicted interdomain linker and corresponds to the first amino acid of ␣-helix 1 (␣1) in the TlyA homology model (Fig. 2F). Crystals of TlyA CTD suitable for structural determination formed within 5 days and diffracted to 1.7 Å resolution.
The TlyA CTD Adopts a Class I Methyltransferase Fold-The structure of TlyA CTD was solved using a TlyA homology model, and unambiguous electron density allowed modeling of amino acids Ser 64 -Pro 268 , producing a final refined model with R work /R free of 0.188/0.218 (also see Table 1). The TlyA CTD structure contains a RrmJ/FtsJ Rossmann-like methyltransferase fold comprising seven ␤-strands (with topology 131211⅐14152716) sandwiched between six ␣-helices (Fig. 3A). The TlyA CTD structure contains the glycine-rich SAM binding motif I GXGXG (17), albeit with the atypical sequence 90 GASTG 94 , containing a central Gly to Ser variation.
The TlyA CTD domain overlays well with other Class I methyltransferases including the archetypical member of the TlyA family, the 2Ј-O-methyltransferase RrmJ, and the DNA C5-methyltransferase HhaI (19,20). Structural superimposition of TlyA-CTD with other class I methyltransferases was used to model the likely location of SAM in the TlyA binding pocket. Although most features of protein secondary structure overlay well between TlyA and RrmJ (PDB code 1EIZ), severe clashes were observed when the RrmJ bound SAM was placed within the TlyA CTD via protein superimposition because of differences in the more variable loops that link the core ␤-strands. In contrast, modeling using the SAM-bound structure of either the HhaI DNA methyltransferase (PDB code 2HMY) or RlmM 2Ј-O-methyltransferase (PDB code 4B17) (21) places SAM into the TlyA binding pocket with no significant clashes (shown for HhaI in Fig. 3B). The modeled SAM is positioned on top of the loop connecting TlyA core strand ␤1 and ␣2, which contains the SAM-binding motif I 90 GASTG 94 . The central Ser residue of an atypical SAM-binding motif of TlyA is oriented toward the modeled SAM and positioned to directly hydrogen bond with the ribose 3Ј-OH. Additionally, by adopting a different rotomeric state, the hydroxyl of residue Thr 93 within this motif would be positioned for interaction with the carboxyl end of the modeled SAM. Finally, the SAM adenine moiety is modeled within a largely hydrophobic pocket on the surface of TlyA comprising the side chains of residues Val 113 , Ala 136 , and Ile 158 ; the backbone of Gly 114 ; and Asn 135 (Fig. 3C). Thus, the TlyA CTD structure possesses the expected features necessary for interaction with the obligatory methyltransferase co-substrate SAM.
We additionally note that the proposed TlyA catalytic residue Asp 154 is positioned adjacent to the transferable methyl group of the modeled SAM (Fig. 3, B and C). Interestingly, two aromatic amino acids, Tyr 115 and Phe 157 , also line the SAM binding pocket but are oriented into the solvent. These residues could play an important functional role in recognizing the rRNA substrate and coordinating the target nucleotide for catalysis as seen with NpmA-30S complex (22).
The Isolated TlyA CTD Protein Does Not Bind SAM-To begin examining TlyA-co-substrate interactions, we used isothermal titration calorimetry (ITC) to compare the binding of SAM and the methylation reaction by-product S-adenosylhomocysteine (SAH) to the full-length enzyme. His-TlyA bound both SAM and SAH with similar affinities in the low micromolar range ( Fig. 4A and Table 2), comparable with other rRNA methyltransferases (23,24). Surprisingly, however, despite retaining a complete class I methyltransferase SAM-binding fold with the expected conserved motifs, the isolated TlyA CTD protein did not exhibit detectable binding of SAM ( Fig. 4B and Table 2). In contrast, GluC-cleaved full-length His-TlyA (His-TlyA GluC ), i.e. the co-purified NTD and CTD fragments (  Table 2). Together, these data indicate that the methyltransferase fold of our TlyA CTD construct is not sufficient for SAM binding, and thus, one or more elements of the amino- terminal 1-63 residues of TlyA must also play a critical role in SAM co-substrate binding.
The RAWV Tetrapeptide Interdomain Linker Is Critical for TlyA CTD-SAM Interaction-To assess the potential contribution of the TlyA NTD to SAM binding, an amino-terminal domain expression construct was created corresponding to residues 1-63 ending with the RAWV tetrapeptide domain linker sequence (NTD RAWV ; Fig. 2F). Expression and purification of the NTD RAWV required an amino-terminal SUMO fusion tag that was removed using the ubiquitin-like protease (Ulp) prior to use in experiments. Using this new construct, we first tested whether the NTD RAWV and CTD proteins interact, recapitulating the retained association of the GluC-derived NTD and CTD fragments of His-TlyA (Fig. 2, D and E). The NTD RAWV and CTD proteins were mixed with 1:1 stoichiometry and applied to a gel filtration column under identical conditions as used previously for the full-length and GluC-cleaved His-TlyA. In contrast to GluC-cleaved His-TlyA, each individual protein domain eluted as a separate peak with no evidence for their direct association (Fig. 5A). Additionally, no binding was detected by ITC when SAM was titrated into in the sample cell containing NTD RAWV /CTD mixture (data not shown). Thus, separate expression of the NTD RAWV and CTD proteins and in vitro reconstitution failed to recapitulate the observed domain association and SAM binding affinity of the GluC-cleaved fulllength His-TlyA protein.
We reasoned that the inability of the separately expressed TlyA domains to interact and bind SAM (either the CTD alone or as an NTD RAWV /CTD mixture) might arise from an inappropriate choice of domain boundary in our expression constructs. Although the NTD and CTD derived from full-length TlyA by GluC cleavage appear to remain strongly associated (Fig. 2, D and E), we determined that dialysis against high salt (1 M NaCl) containing buffer and subsequent application to the gel filtration column was sufficient to isolate a sample highly enriched for TlyA CTD GluC (Fig. 5B). This CTD GluC bound SAM with an affinity essentially identical to full-length His-TlyA despite depletion of the NTD fragment ( Fig. 5C and Table  2), indicating that the majority of the NTD is dispensable for TlyA interaction with co-substrate. We therefore prepared a new TlyA CTD expression construct corresponding to the precise fragment produced by GluC at the predicted Glu 59 cleavage site, thus placing the RAWV tetrapeptide interdomain linker sequence on the amino terminus of the CTD (residues 60 -268, RAWV CTD; Fig. 2F). Remarkably, addition of the RAWV sequence in RAWV CTD restored wild-type SAM binding affinity to the isolated domain protein ( Fig. 5D and Table 2). Thus, the RAWV tetrapeptide sequence appears essential for SAM binding in TlyA. Given the predicted location of the RAWV sequence in the region linking the two domains of TlyA, we next evaluated the possibility that this tetrapeptide motif might also contribute to TlyA domain interaction only when present on the CTD. An additional construct was therefore generated corresponding to the amino-terminal 1-59 amino acids of TlyA (NTD; Fig. 2F) and the purified, tag-free NTD/ RAWV CTD domain proteins mixed and applied to the gel filtration column as before. Although SDS-PAGE analysis revealed less than stoichiometric association, some NTD protein was found to co-elute with RAWV CTD, in contrast to the alternate fragment combination NTD RAWV /CTD (compare Fig. 5, E and A, respectively). We speculate that the weaker association observed for the separately expressed NTD/ RAWV CTD domain mixture compared with the GluC-cleaved protein likely arises because of the need to accommodate, within the protein-protein interface, an additional Gly-Ser dipeptide on RAWV CTD arising from the thrombin cleavage site used to remove its amino-terminal His tag. Additionally, given that the isolated NTD protein required an amino-terminal SUMO fusion for soluble expression, the Ulpcleaved TlyA NTD may also be heterogeneously folded compared with the native domain derived from full-length TlyA. Nonetheless, although not fully recapitulating the domain complex stability following cleavage with GluC, this result suggests that the RAWV tetrapeptide linker may play a role in association and coordination of the TlyA domains, in addition to being essential for SAM binding.
Trp 62 and Val 63 Are the Most Critical Residues for SAM Binding-BLAST search and multiple sequence alignment of the RAWV CTD sequence in UniProt revealed that the tetrapeptide sequence is strongly conserved in the top 250 TlyA homologs. In particular, position 62 is most highly conserved as a tryptophan, and position 63 is invariant as either a valine or alanine (Fig. 6A). Proteins identified as TlyA homologs include TlyA 2Ј-O-methyltransferases, RrmJ methyltransferases, cytotoxins/hemolysins, cytochrome c oxidase subunit II, and TlyA family members. Similar analysis of all SAM-binding proteins within this set of homologs (i.e. either TlyA or RrmJ methyltransferases) revealed an almost identical pattern of sequence conservation as for all 250 proteins (Fig. 6B). In contrast, among Mycobacterial methyltransferases and hemolysins, Arg 60 was more conserved, and Trp 62 /Val 63 were invariant. TlyA homologs from all other species were more variable with valine and proline conserved at position 60 and alanine conserved at position 63 (Fig. 6, C and D). Finally, among six functionally characterized TlyA methyltransferases with overall identities of 38 -100% to Mtb TlyA (25), Val 63 was found to be invariant, with either a tryptophan or tyrosine present at position 62 (Fig.  6E). Based on this conservation, we predicted Arg 60 and, in particular, Trp 62 and Val 63 may play important roles in TlyA function.
To begin experimentally testing the impact of the RAWV tetrapeptide sequence on the TlyA-SAM interaction, individual single amino acid substitutions were made of each at the four residues within full-length TlyA. Each variant was expressed and purified similarly to the wild-type protein, and CD spectroscopy was used to confirm that none of the substitutions resulted in gross changes to the protein fold (data not shown). The SAM binding affinity of each variant was then measured by ITC as before (Table 3). Arg 60 substitution with  either Ala or Glu modestly impacted SAM binding affinity (ϳ3-fold decrease), whereas an A61V variant had wild-type affinity for the co-substrate. In contrast, substitution of Trp 62 with Phe or Ala resulted in ϳ4and 10-fold reduction in SAM binding affinity, confirming a more significant role for Trp 62 in SAM binding and suggesting that the aromatic nature of the side chain is important given the lesser impact of the Trp to Phe substitution. Finally, the most pronounced decrease in SAM binding affinity was observed for the V63A variant (20-fold), pointing to a critical role for this hydrophobic residue in SAM binding. Structural Plasticity of the RAWV Motif-Because Trp 62 and Val 63 are not predicted by our TlyA CTD structure or the TlyA homology model to interact with SAM, we next wanted to determine their local structural environments to assess how they might stabilize amino acids that directly contact SAM. Two different crystallization conditions were identified for TlyA RAWV CTD that produced distinct crystal forms with the same space group and similar cell dimensions but that differed slightly in their packing within the crystal lattice. In both crystal forms of RAWV CTD, the presence of a symmetry-related molecule near the SAM binding site precluded obtaining a structure of SAM-bound RAWV CTD via soaking of preformed crystals with SAM, and efforts to complex crystals by direct co-crystallization were unsuccessful. Nevertheless, the two structures of RAWV CTD offer further insight into the potential molecular mechanism by which the RAWV motif influences SAM binding. Although the core methyltransferase fold in the two structures is essentially identical (aligning with 0.27 Å root mean square deviation for 160 residues), the structure and position of the RAWV motif varies significantly, adopting an unstructured loop in one crystal (form 1 loop) and an extension of ␣1 of the methyltransferase domain in the second (form 2 helix; Fig. 7, A and C).   In the form 1 loop, electron density allowed modeling of Trp 62 and Val 63 peptide backbone and also the Trp 62 side chain (Fig. 7B). In the form 2 helix, clear density was observed for the peptide backbone of the RAWV motif, as well as side chains of residues Ala 61 -Val 63 (Fig. 7D). In the form 2 helix structure, extension of ␣1 by the RAWV tetrapeptide sequence positions Trp 62 to interact with the backbone of Lys 254 and Gly 255 of a symmetry-related molecule. This arrangement is similar to that of the CTD structure (which lacks Trp 62 ) in which an extended ␣1 would be accommodated, if the RAWV sequence were present, without clashing with a symmetry-related molecule. In contrast, the altered crystal packing of the form 1 loop restricts the ability of the RAWV sequence to extend ␣1. Strikingly, the RAWV sequence instead adopts the same structure as observed for the corresponding region (sequence LRYV) in hemolysin proteins from Streptococcus thermophilus (PDB code 3HP7) and Lactococcus lactis (PDB code 3OPN) with Trp 62 overlaying with Tyr 62 and Tyr 36 , respectively, and stabilized by a hydrophobic interaction with Val 99/99/73 (TlyA/3HP7/3OPN; Fig.   7E). Although detailed interpretations are potentially complicated by the influence of crystal packing contacts on the position of the RAWV CTD amino terminus residues, the two structures nonetheless reveal that the Mtb TlyA RAWV motif is capable of adopting two strikingly different conformations. We speculate that these two conformations might reflect an important functional transition in TlyA; for example, if the relative orientation of the NTD and CTD is altered by interaction with ribosomal subunit substrate. An important final question, however, is whether one or both of the observed conformations of the RAWV motif can provide a suitable mechanistic explanation for its contribution to SAM affinity.
Comparison of the two RAWV CTD structures and the CTD structure reveals changes in the regions surrounding the SAM binding pocket in addition to those in the RAWV sequence structure itself (Fig. 7F). In the ␣-helical conformation, Trp 62 is rotated by 180°from its position in the form 1 loop structure. On the opposite side of the SAM binding pocket, a shift in the peptide backbone is also observed for the loops containing Thr 134 and Tyr 115 , with movements of 3.1 and 3.9 Å for their C␣ atoms, respectively, upon comparison of the form 1 loop with the form 2 helix structure. Because Tyr 115 interacts with symmetryrelated molecules in each structure, we cannot eliminate the possibility that this structural change is influenced in part by crystal packing. However, it is noteworthy that although the Tyr 115 backbone moves away from the SAM molecule, its side chain is reoriented closer to the SAM pocket such that it could contribute to positioning of SAM or the target nucleotide in the TlyA active site.
The most striking differences between the two RAWV CTD structures surround Val 63 , the residue most critical for SAM affinity. In the extended ␣1 of the form 2 helix structure, Val 63 is shifted 4.4 Å toward Thr 93 of the SAM binding motif I (Fig.  7G). Additional small differences are seen in the positions of both Thr 93 and Ser 92 C␣, shifted 1.4 and 1.9 Å, respectively, toward the expected position of the bound SAM. We also note that in both the form 1 loop and, to a lesser extent, the CTD structures, the Thr 93 side chain is oriented with its hydroxyl group oriented away from the SAM pocket (Fig. 7H). In contrast, in the form 2 helix structure, the Thr 93 side chain is reoriented with its hydroxyl group pointing into the SAM pocket and positioned to interact directly with the co-substrate carboxylate group (Fig. 7I).
To evaluate the direct contribution of Thr 93 to SAM binding by TlyA, we measured the SAM binding affinity of a T93A variant. TlyA T93A bound SAM with ϳ3-fold lower affinity compared with the wild-type enzyme ( Table 3), suggesting that Val 63 does not exert its effect exclusively by reorienting Thr 93 for SAM binding. Instead, Val 63 must play a more general role in optimally organizing the SAM-binding pocket that underpins the unexpected contribution of the RAWV motif, and this residue in particular, to TlyA-SAM binding affinity.

Discussion
In the present study we have shown that the TlyA methyltransferase contains a class I Rossmann-like methyltransferase fold with a complete SAM-binding motif. However, the methyltransferase domain (amino acids 64 -268) is not competent for SAM binding, and we identified an additional tetrapeptide motif RXWV as essential for SAM interaction with the methyltransferase domain. In particular, the final two amino acids, Trp 62 and Val 63 , have the most pronounced impacts on SAM affinity. Our structural studies illustrated a role for these amino acids in organizing the conserved GXGXG SAM-binding motif I when they are part of an extended ␣ helix1 of the TlyA methyltransferase domain.
Whether the RAWV motif is an important determinant of SAM binding when present in methyltransferases other than TlyA is an open question. To date, the structures of two other methyltransferases with similar sequences have been determined: RrmJ (RAW 36 X) and human fibrillarin (RAW 138 X). In fibrillarin, any contribution of the RAWX sequence to SAM binding would be indirect as the motif is not part of the first ␣-helix of the methyltransferase domain. However, in contrast to both fibrillarin and TlyA, the RAWX sequence of RrmJ is located in the ␣-helix, which precedes the first ␤-sheet of the methyltransferase domain, and the side chain of Ala 137 defines one part of the SAM binding pocket. The potentially direct influence of the RAWX sequence on co-substrate binding by RrmJ is thus in contrast to the indirect influence we have identified in TlyA.
Our structures and functional data suggest that formation of an extended ␣1 helix by the RAWV motif, and in particular, the movement of Val 63 is necessary to promote formation of an optimal SAM binding pocket in TlyA. However, the question remains why TlyA should require this additional motif outside the conserved SAM-binding fold. Characterized TlyA orthologs (Fig. 6E) have both high conservation of the Trp 62 / Val 63 dipeptide of the RXWV motif and an atypical SAM-binding motif, 90 GASTG 94 . Thus, one possibility is that TlyA may exploit the RAWV motif to compensate for the effects of a SAM binding motif I divergent from the canonical GXGXG sequence. In other homologs that also deviate from the GXGXG motif, such as RrmJ ( 59 GAAPG 63 ) and RlmM ( 219 GACPG 223 ) (19,21), the larger side chain is accommodated through its orientation away from the bound SAM molecule, allowing the backbone carboxyl group to be positioned within hydrogen bonding distance of the SAM 3Ј-OH. In contrast in TlyA, the bulkier Ser 92 points toward the SAM binding pocket, potentially either creating or disrupting interactions important for SAM binding. Our structures suggest that the formation of an extended helical structure and repositioning of Val 63 within ␣1 promotes movement of residues Ser 92 and Thr 93 toward the SAM 3Ј-OH and carboxylate, respectively. These changes induced by Val 63 thus appear to optimally organize the SAM binding pocket and underpin the requirement of the RXWV motif in SAM binding by TlyA.
The RXWV motif connects the methyltransferase domain to the TlyA amino-terminal domain, which is predicted to adopt an S4 ribosomal protein binding fold (16) and likely plays a critical role in rRNA recognition (25). However, precisely how TlyA recognizes its two different nucleotide targets, 16S rRNA C1409 and 23S rRNA C1920, remains to be elucidated. Individual fully assembled 30S and 50S ribosomal subunits are the optimal TlyA substrates (25). Although both C1409 and C1920 reside within RNA helical structures with similar sequences, their overall structural context differs significantly. As such, TlyA must accurately recognize two structurally different substrates. C1409 is located in a region of h44, which packs near multiple 16S rRNA helices to form a complex RNA tertiary surface in the assembled 30S subunit, whereas C1920 is contained within a stem-loop (23S rRNA H69) that protrudes from the surface of the free 50S subunit (26). Could the structural plasticity we observe in the TlyA interdomain linker (RXWV tetrapeptide) also play a role in TlyA recognition of its two substrates? A mechanism of this type has been described for the tRNA G37 methyltransferase TrmD, in which an interdomain linker transitions from being structurally disordered to helical upon substrate binding (27). To begin exploring this idea, we manually appended the TlyA NTD homology model onto each of our two TlyA RAWV CTD structures followed by geometry minimization to reveal the potential for the NTD to be oriented in two very different ways (Fig. 8). On the form 1 loop structure, the NTD is more loosely associated with the CTD and in a similar orientation to hemolysin structures and the full-length TlyA homology model, which is based on a hemolysin template (16). In contrast, modeled on the form 2 helix structure, the NTD is significantly repositioned and packs more closely against the TlyA CTD surface surrounding the SAM binding pocket, more consistent with our observation that the domain fragments from GluC-cleaved His-TlyA remain tightly associated. From this preliminary modeling, we speculate that TlyA may employ a mechanism in which specific recognition via the NTD of its two different substrates may be mediated via a common conformational change in the RXWV interdomain linker. In future studies to test this idea, it would also be interesting to determine whether GluC-cleaved or reconstituted NTD/ RAWV CTD TlyA proteins are able to efficiently methylate ribosomal subunits. Substitutions of some positively charged residues in the TlyA NTD are known to have differential impacts on the activity of TlyA against the 30S and 50S substrates (25). Thus, an appealing feature of a TlyA substrate recognition mechanism exploiting the structural plasticity of the RXWV motif is that interactions made by different regions of the amino-terminal S4 domain, with either 30S or 50S, could lead to a common signal via the interdomain linker for correct substrate recognition and activation of methyltransferase activity.
In summary, the present study has revealed the unexpected but critical importance of the RXWV tetrapeptide motif in the TlyA interdomain linker for SAM binding. Our structures of the RAWV CTD and modeling further suggest a potential role for the structural plasticity of the RXWV motif in regulating communication between the TlyA domains and in specific substrate recognition. Further structural studies of TlyA and its complexes with both 30S and 50S subunits are next needed to fully understand the molecular details of specific substrate recognition and the role of the RXWV motif in the activity of this important antibiotic resistance-associated enzyme.

Experimental Procedures
TlyA Construct Design and Site-directed Mutagenesis-An E. coli codon optimized gene encoding TlyA (Rv1694; UniProt P9WJ63) from Mtb (strain ATCC 25618/H37Rv) was obtained by chemical synthesis (GeneArt) and subcloned into a modified pET44 plasmid (28) for expression of protein with a thrombincleavable aminoterminal hexahistidine tag (His-TlyA). Screening for proteolytic fragments of His-TlyA suitable for structural studies with the Proti-Ace kit (Hampton Research) identified the endopeptidase GluC (Staphylococcus aureus Protease V8) as producing two stable domain fragments, and this observation was used as a guide to produce additional domain constructs (see "Results" for details). Plasmids encoding His-CTD (amino acids 64 -268), His-RAWV CTD (amino acids 60 -268), and full-length His-TlyA with single amino acid substitutions were generated using a whole plasmid PCR protocol (29). Constructs for expression of amino-terminal domain proteins SUMO-NTD (amino acids 1-59) and SUMO-NTD RAWV (amino acids 1-63) were generated by PCR amplification of the corresponding coding region in pET44-His-TlyA using primers containing BsaI and XbaI sites for ligation of the amplicon into the pE-SUMOpro vector (LifeSensors).
TlyA Protein Expression and Purification-E. coli BL21 (DE3) cells transformed with protein-encoding plasmid were grown at 37°C in terrific broth supplemented with ampicillin (100 g/ml) to mid-log phase (A 600 ϭ ϳ0.4 -0.6), induced with 0.5 mM isopropyl ␤-D-1-thiogalactopyranoside, and grown for an additional 3-4 h. Following harvesting by centrifugation, the cells were resuspended in 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole and lysed by sonication. The resulting soluble fraction was applied to a His-Trap HP column (GE Healthcare) equilibrated in the same buffer. The column was washed with 10 column volumes of 50 mM sodium phosphate (pH 8.0) buffer containing 300 mM NaCl and 20 mM imidazole and subsequently eluted with the same buffer, but containing 250 mM imidazole. Further purification was accomplished using a Superdex 75 16/60 gel filtration column (GE Healthcare) equilibrated in gel filtration buffer (20 mM Tris, pH 8.0, 10 mM magnesium acetate, 250 mM ammonium chloride, 6 mM ␤-mercaptoethanol, and 10% glycerol). All domain proteins and sequence variants were expressed and purified in the same way as full-length His-TlyA.
For removal of the amino-terminal hexahistidine tag, protein was mixed overnight with thrombin (Sigma-Aldrich; 5 units/1 mg of fusion protein) and passed over tandem HiTrap benzamidine FF and HisTrap HP columns (GE Healthcare). To remove the His-SUMO tag, His-SUMO-NTD and His-SUMO-NTD RAWV were incubated with Ulp and passed over a HisTrap HP column. After tag cleavage and initial purification step, cleaved TlyA or TlyA domains were concentrated and purified in a second gel filtration chromatography step (as described above).
CD Spectroscopy-CD spectroscopy was performed for fulllength wild-type His-TlyA and variants with single amino acid substitutions in the RAWV tetrapeptide sequence on a Jasco J810 spectropolarimeter using solution conditions and instrument settings as described previously (30). Spectra (260 -190 nm) were collected at 20°C. Averaging and background correction were performed using the Spectra Manager software provided with the instrument and analysis of TlyA secondary structure was accomplished using the CDSSTR deconvolution algorithm via Dichroweb (31).
RT Analysis of 16S and 23S rRNA Methylation-Methylation of 30S and 50S was determined using RT assays with E. coli MRE600 30S and 50S subunits purified as described previously (25,32). In brief, E. coli cells were lysed using a French press, and the 50S and 30S ribosomal subunits fractionated by sucrose gradient centrifugation and individually isolated by pelleting of