Active site mapping of yeast aspartyl-tRNA synthetase by in vivo selection of enzyme mutations lethal for cell growth.
The active site of yeast aspartyl-tRNA synthetase has been characterised by structural and functional approaches. However, residues or structural elements that indirectly contribute to the active site organisation have still to be described. They have not been assessed by simple analysis of structural data or site-directed mutagenesis analysis, since rational targetting has proven difficult. Here, we attempt to locate these functional features by using a genetic selection method to screen a randomly mutated yeast AspRS library for mutations lethal for cell growth. This approach is an efficient method to map the active site residues, since of the 23 different mutations isolated, 13 are in direct contact with the substrates. Most of the mutations are located in a 15 A radius sphere around the ATP molecule, where they affect the very conserved residues of the class-defining motifs. The results also showed the importance of the dimer interface for the enzyme activity: a single mutation of the invariant proline residue of motif 1 led to a structural defect inactivating the enzyme. From in vivo complementation studies it appeared that the enzyme activity can be recovered by reconstitution of an intact interface through the formation of heterodimers. We also show that a single mutation affecting an interaction with G34 of the tRNA can inactivate the enzyme by inducing a relaxation of the tRNA recognition specificity. Finally, several mutants whose functional importance could not be assessed from the structural data were selected, demonstrating the importance of this type of approach in the context of a structure-function relationship study.
D019943:Amino Acid Substitution; D000926:Anticodon / Q000378:metabolism; D001226:Aspartate-tRNA Ligase / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002455:Cell Division; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D005800:Genes, Fungal; D005804:Genes, Lethal; D008958:Models, Molecular; D016296:Mutagenesis; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D012349:RNA, Transfer, Asp / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D012641:Selection, Genetic; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity