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Leucyl-tRNA synthetase (LeuRS) is a multidomain enzyme that catalyzes Leu-tRNALeu formation and is classified into bacterial and archaeal/eukaryotic types with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNALeu through different modes of interaction. In the human pathogen Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNASer (CatRNASer(CAG)) in addition to its cognate CatRNALeu, leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNASer, suggesting the significance of their highly divergent CTDs in tRNASer recognition. The results of this study provided the first evidence of the indispensable function of the CTD of eukaryotic LeuRS in recognizing non-cognate CatRNASer and cognate CatRNALeu. Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in the leucylation process: mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knock-out strain.
Aminoacyl-tRNA synthetases (aaRSs)3 are a family of enzymes that catalyze aminoacyl-tRNA formation, playing a pivotal role in protein translation (1). In general, this catalytic process occurs in two steps. First, the amino acid is activated by ATP to form an aminoacyl-adenylate (aa-AMP) intermediate; second, the activated amino acid is transferred to the CCA terminus of the cognate tRNA to form aminoacyl-tRNA (1, 2). Based on the conserved sequences and properties of structural motifs, the 20 aaRSs that account for this catalytic process are divided into two classes (3, 4). Class I aaRSs utilize a Rossmann fold (characterized by “HIGH” and “KMSKS” motifs) to perform their tRNA-charging activities (5). Class II aaRSs dimerize to bind ATP and amino acids through an antiparallel β-fold catalytic site with signature motifs (3, 4). Leucyl-tRNA synthetase (LeuRS), which is a class I aaRS, is further classified into bacterial and archaeal/eukaryotic types on the basis of CP1 domain insertion site and orientation (6,–8). Both types of LeuRS possess a catalytic domain (for amino acid activation and tRNA charging), a CP1 domain (for editing), an α-helix bundle domain, and a C-terminal domain (CTD; for tRNA binding) (9). However, the two types of LeuRS show divergence in the primary sequence and tertiary folding of the CTD. The CTD of bacterial LeuRS is compacted into an αβ domain surrounding by a four-stranded β-sheet (7), and that of archaeal LeuRS comprises a three-stranded β-sheet surrounded by α-helices (10). Seryl-tRNA synthetase (SerRS) belongs to class II aaRSs and is a homodimer, which is distinct from LeuRS in structure and the tRNA recognition mechanism (11, 12).
Candida albicans, a human fungal pathogen, has an ambiguous codon deciphering mechanism in which the universal leucine codon CUG is decoded as both Ser (97%) and Leu (3%) during ribosome translation, leading to proteome ambiguity (11). This ambiguity may create novel protein functionalities to speed up evolution or block sexual reproduction (13). Interestingly, it is a uniquely evolved C. albicans tRNASer(CAG) (CatRNASer(CAG); CatRNASer) that mediates this codon reassignment with no tRNALeu(CAG) isoacceptor existing (14). CatRNASer(CAG) is a chimeric tRNA containing the main body (including acceptor stem, D-stem/loop, TψC stem/loop, and long variable stem/loop) of tRNASer and anticodon stem/loop of tRNALeu (Fig. 1A) (14,–16). Both yeast tRNASer and tRNALeu are class II tRNAs, which have a long variable arm and share similar L-shaped tertiary structures (17,–19). Therefore, CatRNASer(CAG) could be recognized by CaLeuRS through the anticodon triplet and methylated G37 (m1G37), which are characteristics of yeast tRNALeu (Fig. 1A), in addition to being recognized by CaSerRS through the (GC)3 helix of the long variable arm and the discriminator G73 of tRNASer (16). However, G33, which distorts the anticodon stem, leads to decreased leucylation efficiency of CatRNASer(CAG) (Fig. 1A) (20, 21). Overall, the elements of CatRNASer(CAG) to be recognized by both CaSerRS and CaLeuRS have been clearly established (16, 20, 21).
CaSerRS, a key aaRS in CUG reassignment, contains only one CUG codon-encoded residue at position 197 in the dimerization interface of the enzyme (22). Replacement of Ser197 by Leu197 in CaSerRS causes a local structure rearrangement and induces slightly higher catalytic activity without affecting Ser activation. The N-terminal catalytic domain of CaSerRS interacts with the long variable arm of tRNASer with its CTD stabilizing the intramonomer interaction (22, 23). Thus, the element in CaSerRS that recognizes tRNASer has been revealed. Another critical aaRS involved in CUG decoding ambiguity, CaLeuRS, with 1,097 residues, also contains a single CUG codon-encoded residue at position 919, located in its CTD (from Gly894 to Glu1097) (24). The two isoforms of CaLeuRS (CaLeuRS-Ser919 and -Leu919) do not differ in amino acid activation activity; however, CaLeuRS-Leu919 has higher efficiency in leucylating CatRNALeu (24). CaLeuRS-Ser919 predominates in vivo; therefore, this isoform is designated as the wild-type (WT) enzyme. For the interaction between CaLeuRS and CatRNASer, the elements in CaLeuRS that recognize CatRNASer(CAG) have not been identified.
On the basis of crystal structures of bacterial and archaeal LeuRSs, the CTD folds into a separated domain and is disordered in the absence of tRNA (7, 25). Indeed, deletion analysis showed that the CTDs of Escherichia coli LeuRS (EcLeuRS), Thermus thermophilus LeuRS (TtLeuRS), Pyrococcus horikoshii LeuRS (PhLeuRS), and Natrialba magadii LeuRS (NmLeuRS) are all indispensable for leucylating tRNALeu (6, 7, 10, 26,–28). However, deletion of the CTD enhanced the leucylation activity of yeast mitochondrial LeuRS, emphasizing its adaptation in RNA splicing (28). Several studies have explored the concrete recognition elements in the CTD of LeuRSs for cognate tRNALeu. In EcLeuRS, Ala or Asp mutations of several conserved residues had only minimal effects on the aminoacylation activity as did the corresponding double and triple sites mutants (28). In another bacterial LeuRS, Mycobacterium tuberculosis LeuRS (MtLeuRS), several residues were shown to maintain the hydrophobic environment to stabilize the conformation of its CTD and to orient the tRNA (27). In addition, the main chains of the C-terminal Pro962 and Glu967 of PhLeuRS are crucial for recognizing the A47c and G47d of PhtRNALeu (10). Our previous study showed that, in addition to CaLeuRS, other eukaryotic LeuRSs, including Saccharomyces cerevisiae LeuRS (ScLeuRS) and Homo sapiens LeuRS (HsLeuRS), can also leucylate CatRNASer; however, both bacterial and archaeal LeuRSs, including EcLeuRS, C. albicans mitochondrial LeuRS (CamtLeuRS), and PhLeuRS, could not (24). In particular, eukaryotic LeuRS and archaeal PhLeuRS only exhibit obvious divergence in their CTD, indicating the potential significance of the CTD of eukaryotic LeuRS in tRNASer(CAG) recognition.
Above all, the only unexplored question concerning interaction between CaLeuRS and CatRNASer(CAG) seems to be identification of the elements in CaLeuRS that recognize CatRNASer. Besides, although the function of the CTD of bacterial and archaeal LeuRSs in recognition of tRNALeu has been studied extensively by structural and biochemical methods, the potential role of the CTD of eukaryotic LeuRSs in the recognition of tRNALeu has never been reported. Here, our results first showed that CTD of CaLeuRS (CaCTD) is indispensible for leucylating both CatRNASer(CAG) and CatRNALeu (CatRNAs). Additionally, three highly conserved lysine residues within CaCTD were identified as important for leucylating both tRNAs in an additive manner both in vitro and in vivo. In combination, our data identified the specific mechanism concerning recognition of CatRNASer(CAG) by CaLeuRS and improved our understanding of the recognition of tRNALeu by eukaryotic LeuRS.
l-Leu, l-norvaline, dithiothreitol (DTT), ATP, CTP, GTP, UTP, 5′-GMP, tetrasodium pyrophosphate, inorganic pyrophosphate, Tris-HCl, MgCl2, NaCl, and activated charcoal were purchased from Sigma-Aldrich. [3H]Leu, tetrasodium [32P]pyrophosphate, and [α-32P]ATP were obtained from PerkinElmer Life Sciences. Pfu DNA polymerase, the DNA fragment rapid purification kits, and the plasmid extraction kits were purchased from Tiangen (China). The KOD-plus mutagenesis kits were obtained from Toyobo (Japan). T4 ligase, nuclease S1, and restriction endonucleases were obtained from Thermo Scientific (Pittsburgh, PA). Ni2+-nitrilotriacetic acid Superflow was purchased from Qiagen Inc. (Germany). Polyethyleneimine cellulose plates were purchased from Merck. Pyrophosphatase was obtained from Roche Applied Science. The dNTP mixture was obtained from Sangon (China). Oligonucleotide primers were synthesized by Biosune Bioscience (Shanghai, China). Competent E. coli Top10 and RosettaTM 2 (DE3) cells were prepared in our laboratory.
The plasmids containing genes encoding CaLeuRS, ScLeuRS, HsLeuRS, and EcTrmD, pET28a-CaleuS (24), pET28a-ScleuS (29), pET22b(+)-HsleuS (30), and pET28a-Ectrmd (24), respectively, were constructed in our laboratory. The plasmid expressing E. coli tRNA nucleotidyltransferase was provided by Dr. Gilbert Eriani (CNRS, Strasbourg, France). Five deletion mutants of the C terminus of CaLeuRS, CaLeuRS-ΔC1 (Glu1097 deleted), -ΔC2 (Val1096 and Glu1097 deleted), -ΔC3 (Asn1095–Glu1097 deleted), -ΔC4 (Lys1094–Glu1097 deleted), and -ΔC5 (Ile1093–Glu1097 deleted); five Ala replacement mutants of the C-terminal residues, CaLeuRS-E1097A, -V1096A, -N1095A, -K1094A, and -I1093A; 15 Ala replacement mutants of the conserved and/or charged residues, CaLeuRS-Y921A, -R923A, -K938A, -K939A, -K940A, -K941A, -K943A, -P963A, -Q966A, -K997A, -K1007A, -R1009A, -R1021A, -P1086A, and -P1089A; five Asp replacement mutants, CaLeuRS-R923D, -K938D, -K941D, -K1007D, and -P1086D; three Gln replacement mutants, CaLeuRS-K938Q, -K941Q, and -K1007Q; three double sites mutants, CaLeuRS-K938D/K941D, -K938D/K1007D, and -K941D/K1007D; and one triple sites mutant, CaLeuRS-K938D/K941D/K1007D, were constructed by gene mutagenesis using pET28a-CaleuS as a template. By DNA recombination, a chimeric PhLeuRS-CaCTD was obtained by replacing the CTD of PhLeuRS (PhCTD, Val815–Asp967) with CaCTD (Gly894–Glu1097). The genes encoding all mutants were confirmed by DNA sequencing (Biosune Bioscience) and ligated into pET28a to obtain the recombinant plasmids, which were transformed into E. coli Rosetta 2 (DE3) cells to produce the proteins. A single colony of each transformant was chosen and cultured in 500 ml of 2× YT medium (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl) at 37 °C. When the cells reached midlog phase (A600 = 0.6), expression of the recombinant protein was induced by the addition of 0.2 mm isopropyl 1-thio-β-d-galactopyranoside for 12 h at 16 °C. Protein purification was performed according to a method described previously (8).
In C. albicans, only CatRNASer(CAG) bears a CAG anticodon, and no tRNALeu(CAG) exists. Therefore, in this study, we used the CatRNALeu(UAA) isoacceptor to measure the leucylation activity of CaLeuRS. pTrc99b-CatRNALeu(UAA) and pTrc99b-CatRNASer(CAG) were constructed in our laboratory (22). Detailed T7 in vitro runoff transcription of CatRNALeu and CatRNASer was performed according to our previously described method (31). The purified CatRNASer transcript was methylated at position G37 by EcTrmD because the methyl group of m1G37 of CatRNASer is a critical element for recognition by CaLeuRS (13, 32). All CatRNASer used in this study refers to m1G37 CatRNASer(CAG). In vitro transcription of HstRNALeu(CAG) was performed as described previously (30). 32P labeling of CatRNALeu or CatRNASer was performed at 37 °C in a mixture containing 60 mm Tris-HCl (pH 8.0), 12 mm MgCl2, 15 μm CatRNALeu or CatRNASer, 0.5 mm DTT, 15 μm ATP, 50 μm tetrasodium pyrophosphate, 0.666 μm [α-32P]ATP, and 10 μm E. coli tRNA nucleotidyltransferase for 5 min. Finally, 0.8 unit/ml pyrophosphatase was added to the mixture for 5 min. Phenol/chloroform extraction and precipitation of [32P]CatRNALeu and [32P]CatRNASer was performed, and the products were dissolved in 5 mm MgCl2.
For the first step of the aminoacylation reaction, amino acid activation was measured by an ATP-PPi exchange assay at 30 °C in a reaction mixture containing 60 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 2 mm DTT, 4 mm ATP, 2 mm [32P]tetrasodium pyrophosphate (22 cpm/mol), 1 mm Leu, and 20 nm CaLeuRS or its variants. Samples of the reaction mixture (9 μl) were removed at 5-min intervals and immediately added to 200 μl of quenching solution (2% activated charcoal, 3.5% HClO4, and 50 mm tetrasodium pyrophosphate) and mixed by vortexing for 20 s. The solution was filtered through a Whatman GF/C filter followed by washing with 20 ml of Milli-Q water and 10 ml of 95% ethanol. The filter was dried, and [32P]ATP was measured using a scintillation counter (Beckman Coulter).
Leucylation of CatRNALeu was performed at 30 °C in a reaction mixture containing 60 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 2 mm DTT, 4 mm ATP, 20 μm [3H]Leu, 10 μm CatRNALeu, and 20 nm CaLeuRS or its variants. Samples of the reaction mixture (9 μl) were removed onto a Whatman filter at 2-min intervals. After washing with 5% trichloroacetic acid (three times) and 95% ethanol (twice), the filters containing precipitated [3H]leucyl-tRNALeu were dried, and radioactivity was quantified using a scintillation counter. Leucylation of CatRNALeu by ScLeuRS and its variant was performed under the same conditions, and that of HstRNALeu(CAG) by HsLeuRS and its variant was performed as described previously (33). The kinetics of CatRNALeu aminoacylation by CaLeuRS and its variants were determined in the presence of varying concentrations of tRNALeu (0.5–32 μm).
Misleucylation of [32P]CatRNASer was carried out at 30 °C in a reaction mixture containing 60 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 2 mm DTT, 4 mm ATP, 40 mm Leu, 5 μm “cold” CatRNASer, 1 μm [32P]CatRNASer, and 1 mm CaLeuRS or its variants. Aliquots (9 μl) were removed at specific time points for ethanol precipitation with sodium acetate (pH 5.2) at −20 °C overnight. The precipitated samples were centrifuged at 12,000 × g at 4 °C for 30 min, dried at room temperature for 30 min, and digested with 6 μl of nuclease S1 (25 units) for 1 h at 37 °C. After treatment with nuclease S1, leucyl-[32P]tRNA should produce leucyl-[32P]AMP, and free [32P]tRNA should produce [32P]AMP. Quenched aliquots (2 μl) of the digestion mixture were spotted on a thin polyethyleneimine cellulose plate and separated by the thin layer chromatography (TLC) in 0.1 m ammonium acetate and 5% acetic acid. Known amounts of [α-32P]ATP were diluted stepwise and spotted onto the plate for quantification. After visualization by phosphorimaging, the data were analyzed using Multi-Gauge Version 3.0 software (Fujifilm). Misaminoacylation of [32P]CatRNASer by ScLeuRS, HsLeuRS, and their variants was performed under the same conditions except that misaminoacylation by HsLeuRS and its variant was performed at 37 °C.
100 nm m1G37 CatRNASer(CAG) or CatRNALeu(UAA) was incubated with a range of CaLeuRS (0–12 μm) or its mutants in 20 μl of buffer (20 mm Tris-HCl (pH 8.0), 100 mm NaCl, and 15% glycerol) on ice for 20 min. After incubation, 1 μl of loading buffer (0.25% bromphenol blue and 30% glycerol) was added to the sample, and then the sample was loaded into a 6% polyacrylamide native gel. The electrophoresis was carried out at 4 °C at a constant voltage of 100 V for 80 min using 50 mm Tris-glycine buffer. The gel was then stained with ethidium bromide. The RNA bands were quantified by using a Fujifilm imaging analyzer.
The yeast leuS gene was amplified by PCR using pET28a-ScleuS as a template, digested with BamHI and EcoRI, and then ligated into the p416GPD plasmid (predigested with BamHI and EcoRI) to generate p416GPD-ScleuS (leuS+, ura+) (Fig. 2). The diploid yeast strain BY4743-ScleuS+/− (Dharmacon), which contains only one chromosomal copy of leuS with the other replaced by a G418 resistance gene, was transformed with the rescue plasmid p416GPD-ScleuS. The transformants were cultured on synthetic dropout medium without uracil (SD/Ura−). Dissection of spores and further genotype confirmation were performed as described previously (34). The spores that grew on the SD/Ura− plate and failed to grow on SD/Ura−/5-fluoroorotic acid plate were selected as the yeast leuS gene knock-out strain, which was designated ScΔleuS (Fig. 2).
To decrease the expression level, the promoter of p425TEF was replaced by that of endogenous ScleuS gene in a stepwise manner. First, the TEF promoter was deleted in one round of mutagenesis. Second, an 800-bp DNA fragment upstream of the yeast leuS open reading frame was amplified and ligated into the vector to obtain reconstructed shuttle vector p425ScPr. The gene encoding CaLeuRS was subcloned from pET28a-CaleuS and then inserted between the PstI and XhoI sites of p425ScPr to obtain p425ScPr-CaleuS. The gene encoding a His6 tag was inserted at the 5′-terminus of CaleuS to obtain recombinant plasmid p425ScPr-His6-CaleuS. Mutations were engineered by site-directed mutagenesis using p425ScPr-His6-CaleuS as a template (Fig. 2). The constructs were introduced into ScΔleuS strain using the lithium acetate method. Transformants were selected on SD/Ura−/Leu− plates, and a single colony of each transformant was cultured in liquid SD/Leu− medium. To compare the growth rate of each transformant, yeast cultures were diluted to a concentration equivalent to A600 = 1, and a 10-fold dilution of the yeast was dropped onto the SD/Leu−/5-fluoroorotic acid plate to induce the loss of the rescue plasmid (p416GPD-ScleuS). Complementation was observed by comparing the growth rates of ScΔleuS expressing CaLeuRS or its different variants.
Yeast transformants were grown in 20 ml of liquid SD/Leu− medium. The yeast were then harvested; resuspended in ice-cold lysis buffer containing 50 mm Tris-HCl (pH 7.5), 100 mm NaCl, 1 mm EDTA (pH 8.0), 1% Triton X-100, 0.1% sodium deoxycholate, 1 mm PMSF, and 1 mm DTT; mixed with a few glass beads; vortexed rigorously for 10 s (three times at 1-min intervals); and then centrifuged at 10,000 × g for 10 min. The supernatant was separated by SDS-PAGE (10% gel) and transferred to a PVDF membrane. The membrane was blocked with PBST (phosphate-buffered saline containing 0.05% Tween 20) with 5% nonfat dried milk for 3 h at room temperature. Membranes were hybridized overnight with anti-His6 antibody (Abmart, M20001) or anti-α-tubulin antibody (Cell Signaling Technology, 3873S) separately at 4 °C. After incubating with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, immunoreactivity was detected using LAS4000 (Fujifilm) and the SuperSignal West Pico Trial kit (Thermo Fisher Scientific). The results were quantified using Multi-Gauge Version 3.0 software. The total amount of protein was normalized according to that of α-tubulin, and the amount of CaLeuRS was designated as 100%.
Our previous study showed that only eukaryotic LeuRSs (e.g. CaLeuRS, ScLeuRS, and HsLeuRS) could charge CatRNASer(CAG); however, archaeal LeuRS (e.g. PhLeuRS) and bacterial/mitochondrial LeuRS (e.g. EcLeuRS and CamtLeuRS) could not (24). The archaeal and eukaryotic LeuRSs appear to be derived from the same ancestor with the most divergence in the CTD, implying that this region may be critical for recognizing CatRNASer. To address this question, we truncated the CTD of CaLeuRS (from Gly894 to Glu1097), based on the crystal structure of PhLeuRS (Protein Data Bank code 1WKB), obtaining a CTD-truncated mutant (CaLeuRS-ΔC) (Fig. 1B) (6). CaLeuRS-ΔC could be purified to high homogeneity and retained intact leucyl-adenylate synthesis activity (data not shown), suggesting that deletion of CTD had no direct effect on the secondary structure of CaLeuRS and the catalytic active site for amino acid activation. However, it totally lacked the leucylation activity for CatRNASer, indicating the indispensable role of the CTD in Leu-CatRNASer formation (Fig. 3, A and B). Similar results were obtained in assays of the leucylation of CatRNALeu by CaLeuRS-ΔC (Fig. 3C), supporting the hypothesis that the CTD was critical for aminoacylation of tRNA. To further determine the importance of CaCTD in leucylating CatRNASer and CatRNALeu, we substituted CaCTD for PhCTD in PhLeuRS to obtain a mosaic enzyme, PhLeuRS-CaCTD. Compared with PhLeuRS, the mosaic enzyme gained the ability to leucylate CatRNASer and CatRNALeu, further indicating the importance of CaCTD in capturing CatRNAs for aminoacylation (Fig. 3, D, E, and F).
PhLeuRS shows high homology with CaLeuRS (Fig. 4A), and simultaneous deletion of its C-terminal Ile966 and Glu967 completely abolishes the leucylation activity (10). Primary sequence alignment showed that Ile1093 of CaLeuRS is highly conserved among yeast and archaeal LeuRSs and corresponds to Ile966 of PhLeuRS (Fig. 4A). To explore whether CaLeuRS interacts with tRNAs in a similar manner to PhLeuRS, we performed progressive deletions from the C terminus of CaLeuRS, obtaining five mutants, CaLeuRS-ΔC1 to -ΔC5, all of which retained intact amino acid activation activity (data not shown). Further data showed that CaLeuRS-ΔC1 and -ΔC2 showed similar leucylation activity to the WT in the presence of CatRNASer; CaLeuRS-ΔC3 and -ΔC4 gradually decreased their leucylation activity, and CaLeuRS-ΔC5 abolished the leucylation activity (Figs. 4B). Similar results were obtained in assays of the leucylation of CatRNALeu by the five deletion mutants (Fig. 4C). However, the substitution of Ala for the five terminal residues (Glu1097, Val1096, Asn1095, Lys1094, and Ile1093) of CaLeuRS showed similar leucylation activities to the native enzyme (data not shown), suggesting that either the interaction mode between CatRNA and residues at the C terminus of CaLeuRS is similar to that of the PhLeuRS-tRNALeu system (10) or the C terminus is essential for maintaining proper conformation (the latter issue was subsequently addressed using the ScΔleuS strain). Overall, the results show that CaCTD is critical for leucylating both CatRNASer and CatRNALeu; however, the specific elements within CaCTD for charging tRNAs remain to be explored.
The above data showed that the C-terminal five residues, especially Ile1093, are important for the leucylation activity of CaLeuRS for both CatRNASer(CAG) and CatRNALeu. Despite PhLeuRS having Ile966 corresponding to Ile1093, it still failed to recognize both CatRNAs, indicating that the terminal five residues of CaLeuRS may act to affect the leucylation activity in a manner different from that of PhLeuRS. Therefore, CaCTD must harbor other specific elements for charging CatRNAs. Phylogenetic analysis of 13 LeuRSs from various yeast species revealed a high degree of conservation in their CTDs. To identify key residues within CaCTD that may interact with tRNA directly, 10 conserved basic residues (Arg923, Lys997, Lys1007, Arg1009, Arg1021, and 938KKKKGK943) were mutated to Ala, and the leucylation activities of these mutants were assayed. Additionally, we substituted the conserved Pro residues (Pro963, Pro1086, and Pro1089), Tyr921, and Gln966, which are either polar or potentially structure-crucial, with Ala (Fig. 5). Among the 15 Ala mutants, only CaLeuRS-R923A, -K938A, -K941A, -K1007A, and -P1086A showed marked decreases in leucylation activity compared with the WT (Table 1). To further investigate their mode of interaction with tRNA, we replaced the five residues with Asp, obtaining CaLeuRS-R923D, -K938D, -K941D, -K1007D, and -P1086D. The residue Arg923 may contribute to maintaining stability/conformation of the enzyme because CaLeuRS-R923D mainly produced inclusion bodies during expression of its gene in E. coli. All the other four mutants retained intact Leu activation activity comparable with the WT (data not shown), indicating that these mutations had no effect on the catalytic active site. In the subsequent aminoacylation reaction, we assayed and compared the kinetic constants of the WT and the mutants (Table 1). CaLeuRS-K938D, -K941D, -K1007D, and -P1086D all showed increased Km values (5.43, 10.95, 12.36, and 12.08 μm, respectively) compared with the WT (2.01 μm), showing that the affinity between the mutants and tRNA was disrupted. In addition, the kcat values of the four mutants decreased to different extents, accounting for 33, 63, 18, and 25% of that of the WT, respectively. Because of the increased Km and decreased kcat values, their catalytic efficiencies (kcat/Km) were approximately 12, 12, 3, and 4% of that of the WT, respectively. Substituting of these three lysine residues with acidic Asp affects the tRNALeu-leucylating activity, indicating that the positive charge of the three lysines may be important during the leucylation process. This hypothesis was confirmed by the data that the Gln mutants (CaLeuRS-K938Q, -K941Q, and -K1007Q) also showed decreased catalytic efficiency (about 53, 29, and 38% of that of the WT, respectively) although not as obvious as that of their corresponding Asp mutants (Table 1). And the Km values of all three Gln mutants increased (3.33, 5.54, and 4.01 μm, respectively), indicating a reduction of the affinity between the mutants and tRNA (Table 1).
To explore whether the residues important for Leu-tRNALeu synthesis also affect Leu-tRNASer formation, we investigated leucylation of CatRNASer by CaLeuRS-K938D, -K941D, -K1007D, and -P1086D. All the mutants showed obvious decreased kobs values (0.95 × 10−4, 0.14 × 10−3, 0.25 × 10−4, and 0.28 × 10−4 s−1) in CatRNASer leucylation activity, representing about 50, 70, 10, and 10% of that of the WT (0.20 × 10−3 s−1), respectively (Fig. 6A). However, their corresponding Ala mutants, CaLeuRS-K938A, -K941A, and -P1086A, showed a subtle decrease in the CatRNASer-charging activity (Fig. 6B), indicating that introduction of negatively charged residues at Lys938, Lys941, and Pro1086 hindered the formation of Leu-tRNASer. However, CaLeuRS-K1007A still lost almost half of the CatRNASer-charging activity (kobs = 0.14 × 10−3 s−1) compared with the WT (Fig. 6B). These results indicated that the basic residues (Lys938, Lys941, and Lys1007) contribute significantly to the leucylation of both CatRNALeu and CatRNASer, affecting the affinity of enzyme for the tRNA and/or the efficiency of transition in the aminoacylation reaction, which further explains their high conservation within the CTD of yeast LeuRSs.
We identified three basic residues, Lys938, Lys941, and Lys1007, that were implicated as tRNA-interacting elements within the CTD based on the positive charge of their side chains. However, none of their Asp mutants completely lost the leucylation activity for CatRNASer and CatRNALeu. To check whether these residues interact with tRNA in an additive or cooperative manner, we combined the K938D, K941D, and K1007D in the form of either double site mutants (CaLeuRS-K938D/K941D, -K938D/K1007D, and -K941D/K1007D) or triple site mutant (CaLeuRS-K938D/K941D/K1007D) to study their aminoacylation properties. All the mutants maintained intact Leu activation activities (data not shown), indicating that the double and triple site mutations did not affect the catalytic active site. However, the aminoacylation activity of CaLeuRS-K938D/K941D was obviously decreased to about 10 and 20% of that of the WT for CatRNASer and CatRNALeu, respectively, and CaLeuRS-K938D/K1007D and -K941D/K1007D were even completely devoid of the ability to charge both CatRNASer and CatRNALeu, emphasizing the additive effect of the three lysine residues in aminoacylation (Fig. 7, A and B).
The reduced aminoacylation activity of these mutants was possibly due to their decreased tRNA binding capacity. To verify this possibility, we performed gel mobility shift assays to compare the dissociation constants (Kd) of the interactions between the WT, CaLeuRS-K938D/K941D, -K938D/K1007D, -K941D/K1007D, or -K938D/K941D/K1007D and tRNAs. A shifted band for the CaLeuRS-tRNASer complex was observed in the presence of 1 μm CaLeuRS or more with a calculated Kd value of 2.7 μm (Fig. 8, A and B). However, under the same conditions, no shifted band was observed for CaLeuRS-K938/941D, -K938D/K1007D, -K941D/K1007D, and -K938D/K941D/K1007D with tRNASer even in the presence of 12 μm enzyme (Fig. 8A), suggesting that the double and triple site mutations disrupted the formation of the enzyme-tRNASer complex. A similar shifted band was observed with tRNALeu with a calculated Kd value of 2.6 μm (Fig. 8, C and D); however, no shifted band was observed for CaLeuRS-K938D/K941D, -K938D/K1007D, -K941D/K1007D, and -K938D/K941D/K1007D with tRNALeu (Fig. 8C). Thus, it is postulated that the decrease or loss of leucylation ability by these mutants may be partially due to the reduction of the tRNA binding ability during catalysis. Furthermore, it is notable that CaLeuRS binds tRNASer and tRNALeu with similar affinities in vitro despite the divergence of the main bodies of tRNASer and tRNALeu, indicating the importance of the anticodon arm/loop in the interaction with CaLeuRS.
The K1007D mutation showed the most severe impact on the leucylation activity. Primary sequence alignment revealed that Lys1007 is highly conserved in eukaryotic LeuRSs, ranging from yeast species such as S. cerevisiae and Candida tropicalis to higher organisms, such as H. sapiens, Mus musculus, and Xenopus laevis. The counterparts of CaLeuRS-Lys1007 are identified as Lys1002 in ScLeuRS and Lys997 in HsLeuRS, respectively (Fig. 9A). To assess any functional conservation of this site, we replaced them with Asp in ScLeuRS and HsLeuRS to obtain ScLeuRS-K1002D and HsLeuRS-K997D, respectively. In the presence of CatRNASer, the kobs value of ScLeuRS-K1002D (0.35 × 10−3 s−1) was about 40% of that of ScLeuRS (0.92 × 10−3 s−1), and that of HsLeuRS-K997D (0.11 × 10−2 s−1) was about 70% of that of HsLeuRS (0.16 × 10−2 s−1) (Fig. 9B). Further assay of their tRNALeu-charging activity showed similar results. The kobs value of ScLeuRS-K1002D (0.03 s−1) was approximately 10% of that of ScLeuRS (0.26 s−1) (Fig. 9C), and that of HsLeuRS-K997D (0.28 s−1) was about 40% of that of HsLeuRS (0.67 s−1) (Fig. 9D). These data showed that the counterparts of CaLeuRS-Lys1007 in ScLeuRS and HsLeuRS are also important for the recognition of both CatRNASer(CAG) and their cognate tRNALeus by CTD, emphasizing the importance of this highly conserved basic residue for the recognition function of CTD.
Previous studies showed that CaLeuRS was able to rescue an ScΔleuS strain because CaLeuRS showed high sequence homology with ScLeuRS (64.9%) and could aminoacylate SctRNALeu efficiently (34, 35). Hence, the ScΔleuS strain is a good tool to test the in vivo effect of residues identified in in vitro screens.
CaLeuRS-Leu919 and CaLeuRS-Ser919 are two isoforms differing at position 919, and CaLeuRS-Leu919 has a higher catalytic efficiency for CatRNALeu in the aminoacylation assay (24). Here, both isoforms supported ScΔleuS strain growth to a similar level (Fig. 10A). However, by comparing steady-state protein levels of CaLeuRS-Leu919 and -Ser919 from yeast transformants containing their genes, we found that the amount of CaLeuRS-Leu919 was only 32% of that of CaLeuRS-Ser919, indicating that the insertion of Ser or Leu at position 919 may affect gene expression or protein stability (Fig. 10, C and D).
The growth of ScΔleuS containing CaLeuRS-ΔC1, -ΔC2, -ΔC3, and -ΔC4 was similar to that containing CaLeuRS-Ser919 (Fig. 10A). However, the growth of ScΔleuS containing CaLeuRS-ΔC5 was severely disrupted (Fig. 10A). Western blotting showed that the genes encoding all the truncated proteins were expressed to a similar level in yeast (Fig. 10, C and D). Thus, the inability of CaLeuRS-ΔC5 to support ScΔleuS viability was probably caused by the total loss of aminoacylation capacity, not by non- or decreased expression. Compared with CaLeuRS-Ser919, the five deletion mutants were expressed at lower levels, suggesting that the terminal five residues may act to stabilize the conformation of CaCTD and affect protein expression.
ScΔleuS strains transformed of CaLeuRS-K938D, -K941D, -K1007D, -P1086D, and -K938D/K941D had growth rates similar to that transformed with CaLeuRS-Ser919 (Fig. 10B), suggesting that the mutants exogenously expressed by a high copy plasmid were sufficient in supporting yeast growth despite their low aminoacylation activity (see “Discussion”). However, the growth of ScΔleuS transformed with CaLeuRS-K938D/K1007D and -K941D/K1007D showed an apparent delay to different degrees compared with that of CaLeuRS-Ser919, especially CaLeuRS-K938D/K1007D (Fig. 10B). The ScΔleuS containing CaLeuRS-K938D/K941D/K1007D could not grow on SD/Leu−/5-fluoroorotic acid plates, suggesting that the triple site mutant had totally lost tRNA binding ability even when present in sufficient amount. All the results indicated the significance of Lys938, Lys941, and Lys1007 in binding tRNA in vivo, which is consistent with the results obtained in vitro.
aaRSs aminoacylate their cognate tRNAs to produce materials for protein biosynthesis. Structural studies have shown that they evolved N-/C-domains appended to the catalytic center to capture tRNAs for efficient and accurate aminoacylation. For example, the N-terminal domain, containing lysine-rich motifs, significantly enhances tRNA binding in yeast aspartyl-tRNA synthetase and mammalian lysyl-tRNA synthetase (36, 37). The CTDs of Staphylococcus aureus isoleucyl-tRNA synthetase, T. thermophilus valyl-tRNA synthetase, and E. coli histidyl-tRNA synthetase are also responsible for recognizing tRNA (38,–40).
Generally, LeuRS are classified into two types (6,–8). In fact, based on their divergent CTDs, they are further classified into three types (bacterial, archaeal, and eukaryotic LeuRSs) (6, 7). CTDs of both bacterial and archaeal LeuRSs are responsible for recognizing tRNALeu as negligible leucyl-tRNALeu synthesis was observed in the CTD-deleted mutants of EcLeuRS, TtLeuRS, PhLeuRS, and NmLeuRS (7, 10, 26, 28). However, their concrete recognition mechanisms are divergent. A yeast three-hybrid system and band shift assay showed that the CTD of the β-subunit of Aquifex aeolicus LeuRS was responsible for binding tRNA both in vivo and in vitro; however, the recognition elements remained unclear (41). Although several conserved, positively charged residues were identified within the CTD of EcLeuRS, none of them provided site-specific interaction with tRNALeu (28). Asp scanning of the MtLeuRS CTD identified several residues (Val914, Leu949, Gln915, and Leu964) that contributed to maintain the hydrophobic environment to orient tRNALeu in the correct aminoacylation conformation (27). By contrast, based on the crystal structure of PhLeuRS-tRNALeu complex (Protein Data Bank code 1WZ2), some residues (Asp845, Ile849, Pro962, Ile964, Ile966, and Glu967) within the CTD of PhLeuRS formed base-specific interactions with tRNALeu mainly through van der Waals interactions or hydrogen bonds (10).
Our present study, for the first time, used CaLeuRS as a eukaryotic LeuRS model, revealing that the CTD from a eukaryotic LeuRS is also indispensable for recognizing tRNALeu. Notably, three basic residues (Lys938, Lys941, and Lys1007) within CaCTD were important during the leucylation process; their positively charged side chains may interact with tRNA, consistent with the Arg921 of bacterial MtLeuRS and Lys961 of archaeal PhLeuRS (10, 27). In addition to the three key lysine residues, the C-terminal five residues of CaLeuRS may maintain the proper conformation of the CTD during leucylation but not interact with tRNALeu directly, which contrasts with archaeal PhLeuRS (10). In conclusion, although CTDs from the three kingdoms are all essential for recognizing tRNALeu, the recognition mechanisms have evolved to be distinct, allowing more specific and efficient discrimination of their different cognate tRNAs.
CaLeuRS is unique because it recognizes two types of tRNAs (cognate CaRNALeu and non-cognate CatRNASer(CAG)) (24). In S. cerevisiae, substituting the anticodon arm of SctRNASer with that of SctRNALeu endowed it with leucine charging capacity, emphasizing that the anticodon arm/loop of SctRNALeu plays a dominant role in recognition by ScLeuRS (19). CatRNASer(CAG) is a naturally chimeric tRNA with the main body of tRNASer and the anticodon of tRNALeu (13, 14). Mutation analysis of tRNA determinants showed that the anticodon loops/arms of both CatRNASer(CAG) and CatRNALeu are crucial for recognition by CaLeuRS (13, 19).
In our study, Lys938, Lys941, and Lys1007 within CaCTD contribute to recognizing both CatRNALeu and CatRNASer(CAG), suggesting a similar interaction mode between the CaLeuRS and CatRNALeu/CatRNASer in the aminoacylation state. In addition, the affinities between the CaLeuRS and CatRNALeu/CatRNASer were similar in vitro. All of these observations were consistent with the mutation analysis of the tRNA determinants that indicated the crucial importance of the anticodon loop/arm of CatRNASer and CatRNALeu in the recognition by LeuRS (13, 19). In addition, both CatRNASer and CatRNALeu belong to class II tRNAs with a long variable arm, sharing similar tertiary structures. Therefore, the hypothesis that CaLeuRS recognizes the two tRNAs using the same elements is reasonable. Although the recognition elements within CaCTD for CatRNASer(CAG) and CatRNALeu are similar, CatRNASer(CAG) may still possess some antideterminants for CaLeuRS, leading to tRNASer being a poorer charging tRNA compared with tRNALeu, contributing to the low abundance of Leu in CUG sites.
Although the key recognition elements of bacterial or archaeal CTD have been elucidated, neither single nor double/triple site mutants lost the aminoacylation activity completely (28). In this study, we identified three lysine residues (Lys938, Lys941, and Lys1007) in CaLeuRS and first verified that their double/triple site mutants had more severe disruption or even destruction of the leucylation activity compared with the single site mutants, differing from that of EcLeuRS (28).
Furthermore, the gel mobility shift assays showed that CaLeuRS-K938D/K941D, -K938D/K1007D, -K941D/K1007D, and -K938D/K941D/K1007D could not form obvious enzyme-tRNALeu shifted bands with incalculable Kd values, indicating the importance of the three lysine residues in binding tRNA. However, in vivo complementation assays showed that the three double site mutants still supported yeast growth to some extent. This was probably caused by the double site mutants also binding yeast tRNALeu in vivo because the Kd value may not be accurately captured/reflected by the gel mobility shift assay, which was measured under limited enzyme concentration. Besides, it is possible that expression of double site mutants by the high copy p425ScPr plasmid would produce excess protein to complement the impaired tRNA binding capacity of the mutants. Hence, despite the fact that the double site mutants showed negligible aminoacylation activity and Kd in vitro, they could still complement yeast growth to varying degrees. However, both the in vitro and in vivo data supported the view that the combination of the triple site mutant (CaLeuRS-K938D/K941D/K1007D) had completely lost the aminoacylation activity, suggesting an additive effect of these lysine residues on leucylating CatRNALeu.
Our research focused on exploring the interaction between CaLeuRS and CatRNASer/CatRNALeu. The in vitro data showed that Lys938, Lys941, and Lys1007 are important for leucylating both tRNAs. As CaLeuRS can complement ScLeuRS in vivo (34), we used an ScΔleuS strain in which the gene encoding LeuRS was deleted to investigate the catalytic function of the various mutants mentioned in vitro.
In C. albicans, CaLeuRS has two isoforms, CaLeuRS-Ser919 and -Leu919, because the CUG codon at position 919 could be decoded by CatRNASer(CAG) as Ser or Leu. CaLeuRS-Ser919 is the main form; therefore, we used it as the wild type in the in vitro aminoacylation assay. However, in the complementation assay, both isoforms were introduced into the ScΔleuS to compare their expression levels and ability to rescue the yeast. In our study, although there was a 68% decrease in the relative expression level of CaLeuRS-Leu919 compared with that of CaLeuRS-Ser919, ScΔleuS transformed with the gene encoding CaLeuRS-Leu919 grew slightly better than that transformed with CaLeuRS-Ser919, which reflects the higher catalytic efficiency of CaLeuRS-Leu919 compared with CaLeuRS-Ser919 (24). This strict regulation of the relative amounts of CaLeuRS-Ser919/CaLeuRS-Leu919 might balance the CUG decoding with CaSerRS-Ser919/CaSerRS-Leu919 as well as relieve the potential effect on phenotypic diversity caused by excessive Leu misincorporation (16). Moreover, mutants of CaLeuRS with low aminoacylation activity (such as CaLeuRS-K938D, -K941D, -K1007D, -P1086D, and -K938D/K941D) complemented the loss of ScLeuRS as well as the native enzyme, suggesting that mutants exogenously expressed from high copy plasmids were sufficient to support yeast growth although with minimal activity. A similar phenotype was also observed in complementation of a yeast threonyl-tRNA synthetase knock-out strain (42).
ScΔleuS containing CaLeuRS-K938D/K1007D and -K941D/K1007D showed a growth-retarded phenotype, and ScΔleuS containing CaLeuRS-K938D/K941D/K1007D showed a lethal phenotype, which further emphasized the significance and additive effect of Lys938, Lys941, and Lys1007 in leucylating tRNA in vivo. In addition, CaLeuRS-K938D/K941D/K1007D and -ΔC5 could not rescue ScΔleuS, which suggests that both the three lysine residues and the terminal five residues are indispensable for leucylation of tRNA through either direct interaction or maintenance of the proper conformation. Therefore, we believe that the CTD acts to recognize tRNA through complex interactions.
C. albicans, an opportunistic pathogen, has evolved a chimeric CatRNASer(CAG) that leads to protein translation ambiguity. The resultant ambiguity at the CTG codon is crucial in the morphological switch and virulence of this pathogen. The elements in CaLeuRS that mediate this crucial genetic code ambiguity were undetermined. In this study, we have provided the first evidence and clarified the interaction mechanism of the indispensable function of the CTD of CaLeuRS in recognizing both CatRNASer and CatRNALeu. Our results deepen our understanding of the mechanism that meditates CTG codon ambiguity in C. albicans and improves our knowledge concerning tRNALeu recognition by eukaryotic LeuRSs.
Q.-Q. J., X.-L. Z., and E.-D. W. designed and analyzed the experiments and wrote the paper. Z.-P. F. assisted in obtaining the tRNA and preparation of figures. Q. Y. and Z.-R. R. assisted in expression of the mutant enzyme. Q.-Q. J. performed all the other experiments. All authors reviewed the results and approved the final version of the manuscript.
We are grateful to Prof. Jiang-Ye Chen (Shanghai Institute of Biochemistry and Cell Biology) for giving us the C. albicans genome. We thank Prof. Jin-Qiu Zhou and Ming-Hong He for technical assistance in construction of the yeast knock-out strain.
*This work was supported in part by National Key Basic Research Foundation of China Grant 2012CB911000, Natural Science Foundation of China Grants 31130064 and 91440204, and Committee of Science and Technology in Shanghai Grants 12JC1409700 and 15ZR1446500. The authors declare that they have no conflicts of interest with the contents of this article.
3The abbreviations used are: