Plant-exclusive domain of trans-editing enzyme ProXp-ala confers dimerization and enhanced tRNA binding

Jun-Kyu Byun; John A Vu; Siou-Luan He; Jyan-Chyun Jang; Karin Musier-Forsyth

doi:10.1016/j.jbc.2022.102255

Plant-exclusive domain of trans-editing enzyme ProXp-ala confers dimerization and enhanced tRNA binding

J Biol Chem. 2022 Sep;298(9):102255. doi: 10.1016/j.jbc.2022.102255. Epub 2022 Jul 12.

Authors

Jun-Kyu Byun¹, John A Vu¹, Siou-Luan He², Jyan-Chyun Jang³, Karin Musier-Forsyth⁴

Affiliations

¹ Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA; Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, USA.
² Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA; Department of Horticulture and Crop Science and Center for Applied Plant Sciences, The Ohio State University, Columbus, Ohio, USA.
³ Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA; Department of Horticulture and Crop Science and Center for Applied Plant Sciences, The Ohio State University, Columbus, Ohio, USA. Electronic address: jang.40@osu.edu.
⁴ Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA; Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, USA. Electronic address: musier-forsyth.1@osu.edu.

Abstract

Faithful translation of the genetic code is critical for the viability of all living organisms. The trans-editing enzyme ProXp-ala prevents Pro to Ala mutations during translation by hydrolyzing misacylated Ala-tRNA^Pro that has been synthesized by prolyl-tRNA synthetase. Plant ProXp-ala sequences contain a conserved C-terminal domain (CTD) that is absent in other organisms; the origin, structure, and function of this extra domain are unknown. To characterize the plant-specific CTD, we performed bioinformatics and computational analyses that provided a model consistent with a conserved α-helical structure. We also expressed and purified wildtype Arabidopsis thaliana (At) ProXp-ala in Escherichia coli, as well as variants lacking the CTD or containing only the CTD. Circular dichroism spectroscopy confirmed a loss of α-helical signal intensity upon CTD truncation. Size-exclusion chromatography with multiangle laser-light scattering revealed that wildtype At ProXp-ala was primarily dimeric and CTD truncation abolished dimerization in vitro. Furthermore, bimolecular fluorescence complementation assays in At protoplasts support a role for the CTD in homodimerization in vivo. The deacylation rate of Ala-tRNA^Pro by At ProXp-ala was also significantly reduced in the absence of the CTD, and kinetic assays indicated that the reduction in activity is primarily due to a tRNA binding defect. Overall, these results broaden our understanding of eukaryotic translational fidelity in the plant kingdom. Our study reveals that the plant-specific CTD plays a significant role in substrate binding and canonical editing function. Through its ability to facilitate protein-protein interactions, we propose the CTD may also provide expanded functional potential for trans-editing enzymes in plants.

Keywords: Arabidopsis thaliana; aminoacyl-tRNA; aminoacyl-tRNA synthetase; bimolecular fluorescence complementation; enzyme kinetics; molecular modeling; plant biochemistry; trans-editing domains; translational quality control in plants.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Alanine* / chemistry
Alanine* / genetics
Amino Acyl-tRNA Synthetases* / chemistry
Amino Acyl-tRNA Synthetases* / genetics
Arabidopsis* / enzymology
Escherichia coli
Plant Proteins* / chemistry
Plant Proteins* / genetics
Proline* / chemistry
Proline* / genetics
Protein Biosynthesis* / genetics
Protein Conformation, alpha-Helical
Protein Domains
Protein Multimerization*
RNA, Transfer* / chemistry

Substances

Alanine
Amino Acyl-tRNA Synthetases
Plant Proteins
Proline
RNA, Transfer
AT1G44835 protein, Arabidopsis

Grants and funding

R35 GM141880/GM/NIGMS NIH HHS/United States