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Characterization of active/binding site residues of peptidyl-tRNA hydrolase using biophysical and computational

Rajkumar Kulandaisamy1, Tushar Kushwaha1, Vikas Kumar1

  • 1Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India.

International Journal of Biological Macromolecules
|May 24, 2020
PubMed

Insights

Peptidyl-tRNA hydrolase (PtH) is essential for bacterial survival. Researchers studied its binding with puromycin to understand its mechanism and design inhibitors for combating bacterial diseases.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Ribosome stalling during mRNA translation produces toxic peptidyl-tRNA.
  • Peptidyl-tRNA hydrolase (PtH) is vital for bacterial survival by hydrolyzing peptidyl-tRNA.
  • PtH inhibitors are potential antibacterial agents, but their development is hindered by a lack of mechanistic understanding.

Purpose of the Study:

  • To investigate the binding mechanism of M. tuberculosis and E. coli PtH with puromycin.
  • To identify key active site residues involved in PtH-puromycin interaction.
  • To provide insights for designing novel PtH inhibitors.

Main Methods:

  • Isothermal Titration Calorimetry (ITC) for binding thermodynamics.
  • Fourier-Transform Infrared (FTIR) spectroscopy and Circular Dichroism (CD) for structural changes.
  • Molecular docking and Molecular Dynamics (MD) simulations for structural analysis.

Main Results:

  • ITC revealed similar thermodynamic parameters but a threefold difference in KD for puromycin binding to both PtH enzymes.
  • FTIR and CD studies indicated alterations in PtH secondary structure upon puromycin interaction.
  • Molecular modeling highlighted conserved puromycin interactions, with water-mediated interactions in M. tb-PtH contributing to higher affinity.

Conclusions:

  • Puromycin binding to M. tuberculosis and E. coli PtH is conserved but shows differences in affinity.
  • Structural insights suggest specific interactions and water mediation in M. tb-PtH binding.
  • This study provides a foundation for structure-based design of PtH inhibitors against bacterial infections.
Keywords:
Circular dichroism (CD) spectroscopy and modelling studiesEscherichia coli (E. coli)Fourier transform infra-red (FTIR) spectroscopyIsothermal titration calorimetry (ITC)Mycobacterium tuberculosis (M. tb)Peptidyl tRNAPeptidyl tRNA analoguePeptidyl tRNA hydrolase (PtH)

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