Gcn2 structurally mimics and functionally repurposes the HisRS enzyme for the integrated stress response

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Protein kinase Gcn2 regulates protein synthesis during amino acid starvation.
• Gcn2 activation involves its histidyl-tRNA synthetase (HisRS) domain, with tRNA binding as a proposed mechanism.
• Ribosomal P-stalk proteins are also implicated as alternative Gcn2 activators.

Purpose of the Study:

• To elucidate the structural basis of Gcn2 activation by tRNA.
• To investigate the role of the HisRS domain in Gcn2's regulatory function.
• To understand how metabolic enzymes are repurposed for novel regulatory roles.

Main Methods:

• X-ray crystallography of the *Chaetomium thermophilum* Gcn2 HisRS domain.
• Structural analysis revealing mimicry of HisRS catalytic and anticodon-binding domains.
• Mutational analysis of conserved residues in yeast Gcn2.

Main Results:

• The Gcn2 HisRS domain structurally mimics both catalytic (CD) and anticodon-binding (ABD) domains of authentic HisRS.
• The Gcn2 HisRS domain lacks aminoacylation activity, indicating repurposing for kinase activation.
• Mutations in key ABD residues or the ABD-CD interface impair Gcn2 function in starved cells.

Conclusions:

• Deacylated tRNAs likely activate Gcn2 by binding to its HisRS domain through mimicry of tRNA binding sites.
• The Gcn2 HisRS domain has been repurposed from its ancestral metabolic function to mediate novel regulatory control.
• This study provides a structural basis for tRNA-mediated Gcn2 activation and enzyme repurposing.