Amino acid binding by the Class I aminoacyl‐tRNA synthetases: Role for a conserved proline in the signature sequence

Abstract

Although partial or complete three‐dimensional structures are known for three Class I aminoacyl‐tRNA synthetases, the amino acid‐binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl‐tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl‐tRNA synthetase whose defect is manifested in an elevated K_m for methionine (Barker, D.G., Ebel, J.‐P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449–457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300‐fold elevation in K_m for methionine and has little effect on either the K_m for ATP or the k_cat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the “signature sequence” of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl‐tRNA synthetases and, in the known structures, it is in the nucleotide‐binding fold as part of a loop between the end of a β‐strand and the start of an α‐helix. The phenotype of the mutant and the stability and affinity for methionine of the wild‐type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C‐terminus of the signature sequence. (The importance of this position in amino acid binding was demonstrated from studies on another closely related Class I synthetase [Clarke, N.D., Lien, D.C., & Schimmel, P., 1988, Science 240, 521–523].) Because the target proline is conserved in Class I enzymes, it is not likely to determine amino acid side‐chain specificity directly. Instead, structural modeling suggests that this proline acts as a wedge to open an amino acid‐binding cleft between adjacent strands of the β‐sheet.