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Structural analysis of the active site architecture of the VapC toxin from Shigella flexneri

Kehan Xu1, Emil Dedic1, Ditlev E Brodersen1

  • 1Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10c, Aarhus C, DK-8000, Denmark.

Proteins
|February 3, 2016
PubMed

Insights

The VapC toxin, a PIN domain ribonuclease, targets initiator tRNA. Structural analysis reveals VapC forms dimers and a Hepes molecule mimics RNA binding in its active site.

Area of Science:

  • Structural Biology
  • Biochemistry
  • Microbiology

Background:

  • VapC toxin is a PIN domain protein from Shigella flexneri virulence plasmid.
  • PIN domain proteins share a conserved fold but low sequence similarity.
  • VapC is a Mg(2+)-dependent ribonuclease targeting initiator tRNA(fMet) in vivo.

Purpose of the Study:

  • To elucidate the structural basis of VapC toxin activity.
  • To characterize the active site of VapC mutants.
  • To understand the mechanism of VapC-mediated tRNA cleavage.

Main Methods:

  • X-ray crystallography of VapC active site mutants (D7A, D7N, D98N).
  • Biochemical assays to confirm ribonuclease activity.
  • Structural comparison with related enzymes.

Main Results:

  • Crystal structures of VapC mutants D7A, D7N, and D98N were determined.
  • VapC consistently forms a dimer in crystal structures and solution.
  • A Hepes molecule in the D98N structure occupies the active site, mimicking an RNA nucleotide.

Conclusions:

  • VapC functions as a dimer.
  • The Hepes molecule provides insights into RNA substrate binding and positioning.
  • Structural data aids in understanding the VapC ribonuclease mechanism.
Keywords:
PIN domainRNaseX-ray crystallographytRNAtoxin−antitoxin

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