Isolated CP1 domain of Escherichia coli leucyl-tRNA synthetase is dependent on flanking hinge motifs for amino acid editing activity.

The fidelity of protein synthesis relies upon the interpretation of genetic information by the aminoacyl-tRNA synthetases (aaRSs) (1, 2). Each of the aaRSs is responsible for aminoacylation of its cognate amino acid to a specific set of tRNA isoacceptor molecules. In a two-step reaction, the enzyme binds ATP and its cognate amino acid to form an activated aminoacyl-adenylate intermediate. In the second step, the amino acid moiety is transferred to the 3′ end of the tRNA. Amino acids that are similar in size, shape, and/or polarity threaten enzyme specificity (3-6). For example, discrimination of amino acids that differ by one methyl group such as isoleucine and valine, was theoretically predicted to yield an error rate that was as high as 1 out of 5 (4). However, a number of aaRSs have evolved amino acid editing mechanisms to clear these types of mistakes (6). Separate active sites for aminoacylation and editing are employed in a ‘double sieve’ model to enhance amino acid selection and discrimination (7, 8). The first ‘coarse’ sieve contains the aminoacylation active site, which activates cognate amino acids along with structurally similar noncognate amino acids. The second ‘fine’ sieve is an editing active site, which hydrolyzes noncognate amino acids that are mischarged, but importantly, excludes the correctly charged amino acids. Leucyl- (LeuRS), isoleucyl- (IleRS) and valyl- (ValRS) tRNA synthetases are homologous enzymes that belong to subclass IA of the aaRSs (9). These enzymes hydrolyze mischarged amino acids using a discrete domain called connective polypeptide 1 (CP1 (10)) (7, 11-13). The CP1 domain is connected to the canonical aminoacylation core via two β-strand linkers (14-16). The inserted protein domain splits the primary sequence of the Rossmann nucleotide binding fold that is responsible for aminoacylation into two halves (17). The isolated CP1 domains of Escherichia coli IleRS and Bacillus stearothermophilus ValRS were cloned, expressed as active fusion proteins, and edited mischarged Val-tRNAIle and Thr-tRNAVal, respectively (18). Likewise, the recombinantly isolated CP1 domain of Aquifex aeolicus LeuRS, which is an αβ heterodimer, hydrolyzes mischarged Ile-tRNALeu independent of the main body of the enzyme (19). In contrast, CP1 domains that were isolated from E. coli LeuRS failed to deacylate mischarged Ile-tRNALeu (13, 19). Surprisingly however, the independent CP1 domain hydrolyzed the Ile-minihelixLeu (19). In this report, amino acid editing of the full-length mischarged tRNALeu by the isolated E. coli LeuRS CP1 domain required chimeric protein insertions or additions from the A. aeolicus LeuRS (19). Based on the X-ray crystal structures and homology models of LeuRS (15, 20-24), we hypothesized that regions outside of the LeuRS CP1 domain were required for tRNA binding in the editing complex to confer hydrolysis. We created a series of CP1 domain fragments from E. coli LeuRS that included varied extensions of the N- and C-terminal β-strand ends. Isolation of each of these recombinant fragments identified active CP1 domain constructs that hydrolyzed mischarged Ile-tRNALeu. We propose that the LeuRS β-strands as well as flexible extensions of the β-strands that have been called “hinges” are important to tRNA binding in the amino acid editing complex of LeuRS.