Journal of Molecular Biology
Solution Structure and Dynamics of Peptidyl-tRNA Hydrolase from Mycobacterium tuberculosis H37Rv
Introduction
In the course of elongation of the peptide chain, on average, 10% of the translation aborts prematurely before reaching the stop codon.1, 2, 3 The peptidyl-tRNAs from stalled ribosomes are released by ribosome recycling factor and elongation factor-G.2, 3, 4 The propensity for fall-off is dependent on the specific tRNA involved, for example the rate of tRNALys fall off is up to 30 times higher than that of tRNAGly.5 Accumulation of peptidyl-tRNA can also result from the expression of short ORFs or minigenes.6, 7, 8 The accumulation of peptidyl-tRNAs thus released is toxic for the cell because it can either impair the initiation of translation or slow protein synthesis due to specific tRNA starvation.9, 10, 11, 12, 13, 14
Peptidyl-tRNA hydrolase (Pth, EC 3.1.1.29) enzyme, encoded by the Pth gene, was first characterized for Escherichia coli, and was shown to be essential for its viability. It cleaves the ester bond between the C-terminal end of the peptide and the 2′-or 3′-hydroxyl of the ribose at the end of the tRNA, of accumulated peptidyl-tRNAs, thereby making free tRNA available for reuse in protein synthesis. A specific mutation in the Pth gene, Pth(ts), leads to G101D substitution in the Pth protein. E. coli mutants harboring this mutation can grow normally at 30 °C but cannot sustain growth at non-permissive temperature of 42 °C.11 Another specific mutation in the Pth gene, Pth(rap), has R133H mutation in the Pth protein. This mutant cannot support vegetative growth of bacteriophage λ.15 The 3D structure of monomeric E. coli Pth protein has been solved by crystallographic analysis at 1.2 Å resolution.16 The protein has a single α/β globular domain having a central mixed β-sheet twisted by 90°, which is sandwiched by five α-helices. The active site resides in a channel that is covered by a loop and short helix α4 and contains the residues H20 (catalytic base) and D93, which are responsible for catalytic activity.17 Another protein that is structurally homologous but functionally unrelated to EcPth is chloroplast RNA splicing 2 (CRS2). Even though residues that are essential for Pth activity are conserved in this protein, it could not support the growth of E. coli Pth(ts) cells.18
Archaeal genomes do not contain a gene homologous to bacterial Pth. The peptidyl-tRNA hydrolase activity in archaea is carried out by a completely divergent group of proteins that are labeled as Pth2. Crystal structures of Pth2 proteins from Homo sapiens and Sulfolobus solfataricus, and the NMR structure of Pth2 from Archaeglobus fulgidis have been determined.19, 20, 21, 22 The Pth2 proteins have a structural fold resembling that of thioredoxin and differ in the architecture of the catalytic site from the bacterial Pth.19 Overall, there is no sequence or structural homology between bacterial Pth and archaeal Pth2.
Eukaryotes contain at least one ortholog each of the bacterial Pth and archaeal Pth2. It has been shown that canonical Pth and Pth2 enzymes are not essential for the viability of Saccharomyces cerevisiae.23 Moreover, enzymatic action of rabbit reticulocytes Pth on peptidyl-tRNA substrates results in peptidyl-AMP and tRNA-CC as the products, signifying that the activity is a phosphodiesterase type, which differs from the canonical bacterial esterase activity.24, 25
Macrolide antibiotics, such as azithromycin, clarithromycin, and erythromycin, constitute an important class of therapeutic agents against respiratory tract infections caused by most non-tuberculous mycobacteria.26, 27 However, Mycobacterium tuberculosis and several other pathogenic mycobacteria have natural resistance against macrolides.28, 29 This resistance is because of poor absorption and methylation of the N6 amino group of adenine 2058 of 23 S rRNA by Erm methyltransferase, which significantly raises the off-rates.30 Interestingly, there is a link between macrolide antibiotics and Pth activity. Macrolide and lincosamide antibiotics accentuate the thermosensitive character of the Pth(ts) mutants by increasing the fall-off of peptidyl-tRNA.31 Conversely, over-expression of plasmid-based wild type Pth in E. coli Pth(ts) cells could overcome thermosensitivity, and could alleviate the toxic effect of macrolide antibiotics up to a concentration of 200 μg/ml.32
The points discussed above can be summarized with regard to the following five criteria supporting Pth as an important target for the development of anti-bacterial agents. (A) Pth is essential for bacteria. (B) Eukaryotes have two structurally distinct enzymes for Pth activity with the archaeal ortholog being an alternative to the bacterial ortholog. (C) Orthologs of archaeal and bacterial Pth are non-essential in yeast, and possibly in higher eukaryotes. (D) Rabbit, and possibly other higher eukaryotes, reticulocytes have proteins with phosphodiesterase activity that hydrolyze peptidyl-tRNAs. (E) Inhibitors of Pth may act synergistically with macrolide antibiotics, which are already used as drugs.
M. tuberculosis H37Rv Pth (MtPth) is encoded by ORF Rv1014c. It consists of 191 amino acids and has a molecular mass of 20455 Da. We have shown that the MtPth protein is constitutively expressed in M. tuberculosis H37Rv cells by probing with anti-MtPth antibodies. We have demonstrated the in vitro and in vivo Pth activity of the protein encoded by Rv1014c gene.32 The X-ray crystal structure of this protein has been determined very recently in three crystal forms at 1.98 Å, 2.35 Å and 2.49 Å resolution.33 MtPth has 52% sequence homology and 36% sequence identity with that of E. coli Pth. Residues corresponding to N10, H20 and D93 of E. coli Pth are conserved in MtPth, and are crucial for its enzyme activity.32
Although the enzyme activity and structures of E. coli Pth and MtPth have been characterized extensively, no small-molecule inhibitors for Pth family of proteins have been discovered so far. Pth from Streptococcus pneumoniae (SpPth) has been the target of conventional high-throughput screening. However, duplicate attempts did not lead to hits.34 Bacterial Pth proteins are monomeric proteins of ∼ 21 kDa molecular mass and are, therefore, suitable for structure determination using multidimensional NMR spectroscopy, and for subsequent NMR-based screening. Molecular modeling shows that there are two proximal binding sites for the binding of small molecules on either side of the H20 residue of E. coli Pth, in the crevice containing the active site. Therefore, a linked fragment approach employing NMR-based screening and X-ray crystal-based recombination can provide potent inhibitors of this family of proteins. With this objective, we have initiated solution NMR studies for several bacterial Pth proteins.
Here, we report the three-dimensional solution structure of MtPth determined by NMR spectroscopy, and the dynamic properties of the enzyme assessed from 15N relaxation measurements. The relaxation data were analyzed by model-free formalism,35, 36, 37, 38 and reduced spectral density mapping analysis.39, 40
Section snippets
Structure determination and statistics
A total number of 2161 nuclear Overhauser effect (NOE)-derived distance restraints, 295 dihedral angle restraints, and 179 hydrogen bond restraints were used for structural calculations using CYANA-1.0.5. An ensemble of 40 superimposed backbone traces, representing the solution structure of MtPth is shown in Fig. 1a. The ribbon representation of structure with the lowest target function and its surface charge potential are shown in Fig. 1b and c, respectively. The input data for structural
Discussion
Here, we report the first solution structure of M. tuberculosis peptidyl-tRNA hydrolase (MtPth), which is also the first solution structure for the bacterial Pth family of proteins. E. coli Pth and Chloroplast RNA splicing 2 (CRS2) protein share 39% and 40% sequence identity with MtPth, respectively. Overall, these structures exhibit good resemblance over topology and tertiary structure. The backbone RMSD of MtPth solution structure when compared to EcPth over 191 residues and CRS2 over 185
Preparation of NMR samples
15N-labeled and 13C,15N-labeled Pth samples for NMR spectroscopy were prepared as described.32 For preparation of 13C,15N-labeled sample in 99.8% (v/v) 2H2O, 500 μL of labeled protein was lyophilized and hydrated with 500 μL of 2H2O. This was equilibrated twice by diluting with buffer (20 mM NaH2PO4(pH 6.1), 50 mM NaCl, 0.1% (w/v) NaN3, 2 mM DTT) prepared in 2H2O and concentrated using an Amicon Centricon membrane filter with a 5 kDa cutoff.
NMR spectroscopy and Data processing
NMR experiments were recorded at 30 °C either on
Acknowledgements
We dedicate this article to Dr. C. M. Gupta, DBT distinguished scientist, Central Drug Research Institute, Lucknow, for establishing the NMR facility for Macromolecular structure at CDRI. We thank the National facility for high-field NMR at TIFR, Mumbai for access to the Bruker 800MHz spectrometer. This work was supported by grants from CSIR Network Project SMM-003, NWP-0038, and from the Department of Biotechnology (DBT), India. P.S.V.S.R.K., P.P.P., and A.M. are recipients of research
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2016, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :Structures of Pth from different bacterial species such as E. coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Mycobacterium smegmatis, Francisella tularensis, Acinetobacter baumannii, Staphylococcus aureus, and Streptococcus pyogenes have been determined. All these structures show similar folds and grossly similar catalytic sites [19–27], which makes bacterial Pth an important and universal drug target for the development of broad spectrum antibiotics. Several current publications on Pth structures have heralded Pth as a potential drug target.
Structural and functional insights into peptidyl-tRNA hydrolase
2014, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :Due to the presence of two consecutive prolines, the rigidity of the backbone chain increases substantially so that the substrate binding channels of MsPth and MtPth are narrower in comparison to that in AbPth, EcPth, FtPth and PaPth (Fig. 7). A comparison of B-factors of this segment in the case of all species indicated that this particular stretch has the highest B-factor in the entire protein chain, which is in accordance to other studies which suggest that this segment is involved in substrate binding [35]. The most important residue in this site is Lys142 which is speculated to form an electrostatic interaction with the CCA tail, which leads to binding of the peptidyl-adenosine moiety inside the active site [32].