The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA

Jay Z Yin; Alexander F A Keszei; Scott Houliston; Frantisek Filandr; Jonah Beenstock; Salima Daou; Julia Kitaygorodsky; David C Schriemer; Mohammad T Mazhab-Jafari; Anne-Claude Gingras; Frank Sicheri

doi:10.1016/j.str.2024.02.021

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA

Structure. 2024 Mar 22:S0969-2126(24)00081-9. doi: 10.1016/j.str.2024.02.021. Online ahead of print.

Affiliations

¹ Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON M5G 1X5, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
² Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, ON M5G 1L7, Canada.
³ Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, ON M5G 1L7, Canada; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada.
⁴ Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 4N1, Canada.
⁵ Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON M5G 1X5, Canada.
⁶ Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
⁷ Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON M5G 1X5, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: sicheri@lunenfeld.ca.

PMID: 38531363
DOI: 10.1016/j.str.2024.02.021

Abstract

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

Keywords: GCN2; cryoEM; eIF2α kinase; integrated stress response; pseudoHisRS; pseudoenzyme; tRNA binding.