Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition.

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• The protein kinase PKR is a key antiviral protein that inhibits protein synthesis.
• PKR activation is triggered by double-stranded RNA binding, leading to dimerization and autophosphorylation.
• Phosphorylation of the translation initiation factor eIF2alpha by PKR halts protein synthesis.

Purpose of the Study:

• To identify mutations that activate PKR independently of its regulatory domains.
• To elucidate the role of the kinase catalytic domain in PKR activation and substrate recognition.
• To define the mechanism of PKR activation and its interaction with eIF2alpha.

Main Methods:

• Site-directed mutagenesis to generate PKR variants.
• In vitro kinase assays to measure autophosphorylation and eIF2alpha phosphorylation.
• Analysis of mutations within the PKR catalytic domain and conserved residues.

Main Results:

• Mutations activating PKR were mapped to a dimerization surface on the catalytic domain.
• Specific mutations on this surface inhibited PKR autophosphorylation and eIF2alpha phosphorylation.
• Mutation of Thr446 impaired eIF2alpha phosphorylation and viral pseudosubstrate binding.
• Helix alphaG within the catalytic domain was identified as crucial for eIF2alpha recognition.

Conclusions:

• Catalytic domain dimerization is a critical step in ordered PKR activation.
• Thr446 autophosphorylation and specific eIF2alpha substrate binding are downstream events.
• The findings provide insights into the molecular mechanism of PKR antiviral activity.