title: Substrate specificity of bacterial prolyl-tRNA synthetase editing domain is controlled by a tunable hydrophobic pocket.
abstract: Aminoacyl-tRNA synthetases catalyze the covalent attachment of amino acids onto their cognate tRNAs. High fidelity in this reaction is crucial to the accurate decoding of genetic information and is ensured, in part, by proofreading of the newly synthesized aminoacyl-tRNAs. Prolyl-tRNA synthetases (ProRS) mischarge tRNA(Pro) with alanine or cysteine due to their smaller or similar sizes relative to cognate proline. Mischarged Ala-tRNA(Pro) is hydrolyzed by an editing domain (INS) present in most bacterial ProRSs. In contrast, the INS domain is unable to deacylate Cys-tRNA(Pro), which is hydrolyzed exclusively by a freestanding trans-editing domain known as YbaK. Here, we have used computational and experimental approaches to probe the molecular basis of INS domain alanine specificity. We show that the methyl side chain of alanine binds in a well defined hydrophobic pocket characterized by conserved residues Ile-263, Leu-266, and Lys-279 and partially conserved residue Thr-277 in Escherichia coli ProRS. Site-specific mutation of these residues leads to a significant loss in Ala-tRNA(Pro) hydrolysis, and altering the size of the pocket modulates the substrate specificity. Remarkably, one ProRS INS domain variant displays a complete switch in substrate specificity from alanine to cysteine. The mutually exclusive aminoacyl-tRNA substrate specificities of the WT and engineered INS domains is consistent with the evolution of two distinct editing domains that function to clear Ala-tRNA(Pro) and Cys-tRNA(Pro) in vivo.
authors: Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio 43210, USA.
authors: Sandeep
authors: S
authors: Kumar
authors: Mom
authors: M
authors: Das
authors: Christopher M
authors: CM
authors: Hadad
authors: Karin
authors: K
authors: Musier-Forsyth
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013293:Enterococcus faecalis / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D029968:Escherichia coli Proteins; D000604:Amino Acyl-tRNA Synthetases