Functional dissection of a predicted class-defining motif in a class II tRNA synthetase of unknown structure.
A core of eight beta-strands and three alpha-helices was recently predicted for the active site domain of Escherichia coli alanyl-tRNA synthetase, an enzyme of unknown structure [Ribas de Pouplana, L1., Buechter, D. D., Davis, M. W., & Schimmel, P. (1993) Protein Sci. 2, 2259-2262; Shi, J.-P., Musier-Forsyth, K., & Schimmel, P. (1994) Biochemistry 26, 5312-5318]. A critical part of this predicted structure is two antiparallel beta-strands and an intervening loop that make up the second of three highly degenerate sequence motifs that are characteristic of the class II aminoacyl-tRNA synthetases. We present here an in vivo and in vitro analysis of 21 rationally designed mutations in the predicted 34-amino acid motif 2 of E. coli alanyl-tRNA synthetase. Although this motif in E. coli alanyl-tRNA synthetase is of a different size than and has only two sequence identities with the analogous motif in yeast aspartyl- and Thermus thermophilus seryl-tRNA synthetases, whose structures are known, the functional consequences of the mutations are explainable in terms of those structures. In particular, the analysis demonstrates the importance of the predicted motif 2 in adenylate formation, distinguishes between two similar, but distinct, predicted models for this motif, and distinguishes between the functional importance of two adjacent phenylalanines in a way that strongly supports the predicted structure. The results suggest that similar analyses will be generally useful in testing models for active site regions of other class II aminoacyl-tRNA synthetases of unknown structure.
Davis
M W
MW
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Buechter
D D
DD
Schimmel
P
P
D000215:Acylation; D000409:Alanine; D000412:Alanine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology
D001120:Arginine; D000412:Alanine-tRNA Ligase; D000409:Alanine