ABSTRACT
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 ß-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.
Subject(s)
Bacterial Proteins/chemistry , Carboxylic Ester Hydrolases/chemistry , Mycobacterium smegmatis/chemistry , RNA, Transfer, Amino Acyl/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Conformation, beta-Strand , Sequence Alignment , Substrate SpecificityABSTRACT
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 ß-sheet consisting of six ß-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)â¼0.2µM). The F-actin binding site is not well formed and analysis of (15)N-relaxation data shows that residues in the ß4-ß5 loop region and C-terminal are relatively flexible, which seems to be a determinant for the low F-actin severing activity of LdCof.
Subject(s)
Actin Depolymerizing Factors/chemistry , Leishmania donovani/metabolism , Protozoan Proteins/chemistry , Actins/chemistry , Animals , Calorimetry , Magnetic Resonance Spectroscopy , Muscle, Skeletal/metabolism , RabbitsABSTRACT
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.
