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Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein

Eun Joo Seo1, Furong Liu, Makiko Kawagishi-Kobayashi

  • 1Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Proceedings of the National Academy of Sciences of the United States of America
|October 31, 2008
PubMed

Insights

The double-stranded RNA activated protein kinase PKR is inhibited by poxvirus proteins. Researchers identified mutations in PKR that resist this inhibition, revealing insights into viral defense mechanisms.

Area of Science:

  • Biochemistry
  • Virology
  • Molecular Biology

Background:

  • The innate immune response involves protein kinase PKR, which inhibits protein synthesis to block viral replication.
  • Poxviruses produce inhibitors, like vaccinia virus K3L, that target PKR by mimicking the eIF2alpha substrate.
  • High expression of human PKR is toxic in yeast, but this is suppressed by K3L coexpression.

Purpose of the Study:

  • To identify mutations in PKR conferring resistance to K3L inhibition using a yeast-based screening assay.
  • To characterize the impact of these mutations on PKR's interaction with K3L and its substrate eIF2alpha.
  • To elucidate the structural basis of PKR-K3L interaction and substrate recognition.

Main Methods:

  • Utilized a yeast expression system to screen for PKR mutants resistant to K3L-mediated growth inhibition.
  • Performed in vitro assays to assess K3L binding affinity and eIF2alpha phosphorylation activity of mutant PKR.
  • Mapped identified mutations to the C-terminal lobe of the PKR kinase domain.

Main Results:

  • Identified 12 mutations in the C-terminal lobe of PKR conferring resistance to K3L inhibition in yeast and in vitro.
  • The PKR-D486V mutation significantly reduced K3L binding affinity (15-fold) without affecting eIF2alpha phosphorylation.
  • Results suggest K3L binds to an extensive face on the C-terminal lobe of the PKR kinase domain.

Conclusions:

  • Subtle mutations in the PKR kinase domain can selectively disrupt pseudosubstrate (K3L) inhibition while preserving substrate (eIF2alpha) phosphorylation.
  • The findings delineate the eIF2alpha-binding site on PKR, providing a structural basis for understanding pseudosubstrate inhibition.
  • Differences in the structural flexibility between K3L and eIF2alpha may explain the paradoxical effects of PKR mutations on inhibitor versus substrate interactions.

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