Anupam Jain

Solution structure and dynamics of peptidyl-tRNA hydrolase from Mycobacterium tuberculosis H37Rv.

Eubacterial peptidyl-tRNA hydrolase is an essential enzyme that hydrolyzes peptidyl-tRNAs that are released into the cytoplasm because of premature termination of translation, expression of minigenes, and action of lincosamide and macrolide antibiotics. This averts the arrest of protein synthesis caused by depletion of free tRNA. Recently, we demonstrated that Mycobacterium tuberculosis peptidyl-tRNA hydrolase (MtPth) is present in the cytosol of mycobacterium and is capable of hydrolyzing peptidyl-tRNA. Here, we present the solution structure of MtPth, which is the first solution structure for this family of proteins. MtPth typically consists of seven-stranded mixed beta-sheet surrounded by six alpha-helices. The backbone dynamics for this enzyme were probed by measuring (15)N relaxation parameters and these were analyzed with model-free formalism and reduced spectral density mapping analysis. Overall, the protein molecule has tau(m) of 9.67+/-0.02 ns. The (15)N relaxation data analysis reveals that while majority of the protein backbone is rigid to motions, a short segment consisting of enzymatically critical residue H22, the loop-helix cover over the active site crevice, and the C-terminal helical hairpin exhibit motions on the milli-to microsecond timescale, all of which are linked to interaction with the substrate peptidyl-tRNA.

Solution structure and dynamics of ADF/cofilin from Leishmania donovani

Leishmania donovani ADF/cofilin (LdCof) is a novel member of ADF/cofilin family. LdCof depolymerizes, but does not co-sediment with, rabbit muscle actin filaments. Its F-actin depolymerizing activity is pH independent. Further, it possesses weak F-actin severing activity. In order to better understand its characteristic properties, we have determined the solution NMR structure of LdCof and have analyzed protein backbone dynamics from (15)N-relaxation measurements. The structure of LdCof possesses a conserved ADF/cofilin fold with a central mixed β-sheet consisting of six β-strands which is surrounded by five α-helices. LdCof structure has conserved G/F-actin binding site which includes the characteristic long kinked α-helix (α3). LdCof binds to rabbit muscle ADP-G-actin with 1:1 stoichiometry (K(d)∼0.2μM). The F-actin binding site is not well formed and analysis of (15)N-relaxation data shows that residues in the β4-β5 loop region and C-terminal are relatively flexible, which seems to be a determinant for the low F-actin severing activity of LdCof.

Solution Structures and Dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans

The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand β6. It was also relatively less flexible with only five residues showing motions on a nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.

Unraveling the stereochemical and dynamic aspects of the catalytic site of bacterial peptidyl-tRNA hydrolase.

Bacterial peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) hydrolyzes the peptidyl-tRNAs accumulated in the cytoplasm and thereby prevents cell death by alleviating tRNA starvation. X-ray and NMR studies of Vibrio cholerae Pth (VcPth) and mutants of its key residues involved in catalysis show that the activity and selectivity of the protein depends on the stereochemistry and dynamics of residues H24, D97, N118, and N14. D97-H24 interaction is critical for activity because it increases the nucleophilicity of H24. The N118 and N14 have orthogonally competing interactions with H24, both of which reduce the nucleophilicity of H24 and are likely to be offset by positioning of a peptidyl-tRNA substrate. The region proximal to H24 and the lid region exhibit slow motions that may assist in accommodating the substrate. Helix α3 exhibits a slow wobble with intermediate time scale motions of its N-cap residue N118, which may work as a flypaper to position the scissile ester bond of the substrate. Overall, the dynamics of interactions between the side chains of N14, H24, D97, and N118, control the catalysis of substrate by this enzyme.

Structural characterization of peptidyl-tRNA hydrolase from Mycobacterium smegmatis by NMR spectroscopy

BACKGROUND Accumulation of toxic peptidyl-tRNAs in the bacterial cytoplasm is averted by the action of peptidyl-tRNA hydrolase (Pth), which cleaves peptidyl-tRNA into free tRNA and peptide. NMR studies are needed for a protein homolog with a complete crystal structure, for comparison with the NMR structure of Mycobacterium tuberculosis Pth. METHODS The structure and dynamics of Mycobacterium smegmatis Pth (MsPth) were characterized by NMR spectroscopy and MD simulations. The thermal stability of MsPth was characterized by DSC. RESULTS MsPth NMR structure has a central mixed seven stranded β-sheet that is enclosed by six α-helices. NMR relaxation and MD simulations studies show that most of the ordered regions are rigid. Of the substrate binding segments, the gate loop is rigid, the base loop displays slow motions, while the lid loop displays fast timescale motions. MsPth displays high thermal stability characterized by a melting temperature of 61.71°C. CONCLUSION The NMR structure of MsPth shares the canonical Pth fold with the NMR structure of MtPth. The motional characteristics for the lid region, the tip of helix α3, and the gate region, as indicated by MD simulations and NMR data, are similar for MsPth and MtPth. However, MsPth has relatively less rigid base loop and more compactly packed helices α5 and α6. The packing and the dynamic differences appear to be an important contributing factor to the thermal stability of MsPth, which is significantly higher than that of MtPth. SIGNIFICANCE MsPth structure consolidates our understanding of the structure and dynamics of bacterial Pth proteins.

Structure, dynamics, and biochemical characterization of ADF/cofilin Twinstar from Drosophila melanogaster

BACKGROUND Twinstar is an ADF/cofilin family protein, which is expressed by the tsr gene in Drosophila melanogaster. Twinstar is one of the main regulators of actin cytoskeleton remodelling and is essential for vital cellular processes like cytokinesis and endocytosis. METHODS We have characterized the structure and dynamics of Twinstar by solution NMR spectroscopy, the interaction of Twinstar with rabbit muscle actin by ITC, and biochemical activities of Twinstar through different biochemical assays using fluorescence spectroscopy and ultra-centrifugation. RESULTS The solution structure of Twinstar shows characteristic ADF-H fold with well-formed G/F-site and F-site for interaction with actin. The structure possesses an extended F-loop, which is rigid at the base, but flexible towards its apical region. Twinstar shares similar dynamics for the G/F-site with C. elegans homologs, UNC-60A and UNC-60B. However, the dynamics of its F-loop are different from its C. elegans homologs. Twinstar shows strong affinity for ADP-G-Actin and ATP-G-Actin with Kds of ~7.6 nM and ~0.4 μM, respectively. It shows mild F-actin depolymerizing activity and stable interaction with F-actin with a Kd of ~5.0 μM. It inhibits the rate of the nucleotide exchange in a dose dependent manner. CONCLUSION On the basis of structure, dynamics, and biochemical activity, Twinstar can be taken to execute its biochemical role by facilitating directional growth and maintenance of length of actin filaments. GENERAL SIGNIFICANCE This study characterizes the structure, backbone dynamics, and biochemical activities of Twinstar of Drosophila, which provides an insight into the regulation of actin dynamics in the member of phylum insecta.

NMR assignment of actin depolymerizing and dynamics regulatory protein from Leishmania donovani

Leishmania donovani cofilin displays low sequence similarity to other mammalian cofilins and also possesses characteristic activity of its own. Determination of its solution structure would facilitate understanding of the molecular mechanism of actin dynamics regulation in this disease causing pathogen.

Role of methionine 71 in substrate recognition and structural integrity of bacterial peptidyl-tRNA hydrolase.

BACKGROUND Bacterial peptidyl-tRNA hydrolase (Pth) is an essential enzyme that alleviates tRNA starvation by recycling prematurely dissociated peptidyl-tRNAs. The specificity of Pth for N-blocked-aminoacyl-tRNA has been proposed to be contingent upon conserved residue N14 forming a hydrogen bond with the carbonyl of the first peptide bond in the substrate. M71 is involved in forming a conserved hydrogen bond with N14. Other interactions facilitating this recognition are not known. METHODS The structure, dynamics, and stability of the M71A mutant of Pth from Vibrio cholerae (VcPth) were characterized by X-ray crystallography, NMR spectroscopy, MD simulations and DSC. RESULTS Crystal structure of M71A mutant was determined. In the structure, the dimer interface is formed by the insertion of six C-terminal residues of one molecule into the active site of another molecule. The side-chain amide of N14 was hydrogen bonded to the carbonyl of the last peptide bond formed between residues A196 and E197, and also to A71. The CSP profile of mutation was similar to that observed for the N14D mutant. M71A mutation lowered the thermal stability of the protein. CONCLUSION Our results indicate that the interactions of M71 with N14 and H24 play an important role in optimal positioning of their side-chains relative to the peptidyl-tRNA substrate. Overall, these interactions of M71 are important for the activity, stability, and compactness of the protein. SIGNIFICANCE The work presented provides original and new structural and dynamics information that significantly enhances our understanding of the network of interactions that govern this enzymes activity and selectivity.