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

Ashish Kabra1, Farheen Fatma1, Salman Shahid1

  • 1Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India.

Biochimica Et Biophysica Acta
|July 6, 2016
PubMed
Abstract

Insights

Mycobacterium smegmatis peptidyl-tRNA hydrolase (MsPth) structure reveals insights into bacterial Pth proteins. MsPth exhibits higher thermal stability than M. tuberculosis Pth due to distinct structural dynamics.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Microbiology

Background:

  • Peptidyl-tRNA hydrolase (Pth) prevents toxic peptidyl-tRNA accumulation in bacteria by cleaving peptidyl-tRNA into tRNA and peptide.
  • Structural and dynamic characterization of Pth homologs is crucial for understanding bacterial protein homeostasis.

Purpose of the Study:

  • To characterize the structure and dynamics of Mycobacterium smegmatis Pth (MsPth) using NMR spectroscopy and molecular dynamics (MD) simulations.
  • To compare the structural and dynamic features of MsPth with those of Mycobacterium tuberculosis Pth (MtPth).

Main Methods:

  • Nuclear Magnetic Resonance (NMR) spectroscopy was employed to determine the 3D structure of MsPth.
  • Molecular dynamics (MD) simulations and NMR relaxation studies were used to analyze protein dynamics.
  • Differential Scanning Calorimetry (DSC) was used to assess the thermal stability of MsPth.

Main Results:

  • The MsPth NMR structure features a central seven-stranded β-sheet surrounded by six α-helices.
  • NMR relaxation and MD simulations indicated rigid ordered regions, with specific dynamics observed in substrate binding loops (gate, base, and lid).
  • MsPth demonstrated high thermal stability with a melting temperature of 61.71°C.

Conclusions:

  • MsPth shares the canonical Pth fold with MtPth, with similar motional characteristics in key regions.
  • MsPth exhibits a less rigid base loop and more compact helices α5 and α6 compared to MtPth.
  • These structural and dynamic differences contribute to MsPth's significantly higher thermal stability, enhancing our understanding of bacterial Pth proteins.
Keywords:
Differential scanning calorimetryMolecular dynamics simulationsNMR spectroscopyPeptidyl-tRNA hydrolase

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