C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes both tRNASer and tRNALeu.
ABSTRACT: Leucyl-tRNA synthetase (LeuRS) is a multidomain enzyme that catalyzes Leu-tRNA(Leu) 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 tRNA(Leu) 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 tRNA(Ser) (CatRNA(Ser)(CAG)) in addition to its cognate CatRNA(Leu), leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNA(Ser), suggesting the significance of their highly divergent CTDs in tRNA(Ser) recognition. The results of this study provided the first evidence of the indispensable function of the CTD of eukaryotic LeuRS in recognizing non-cognate CatRNA(Ser) and cognate CatRNA(Leu). 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.
Project description:Aminoacyl-tRNA synthetases should ensure high accuracy in tRNA aminoacylation. However, the absence of significant structural differences between amino acids always poses a direct challenge for some aminoacyl-tRNA synthetases, such as leucyl-tRNA synthetase (LeuRS), which require editing function to remove mis-activated amino acids. In the cytoplasm of the human pathogen Candida albicans, the CUG codon is translated as both Ser and Leu by a uniquely evolved CatRNA(Ser)(CAG). Its cytoplasmic LeuRS (CaLeuRS) is a crucial component for CUG codon ambiguity and harbors only one CUG codon at position 919. Comparison of the activity of CaLeuRS-Ser(919) and CaLeuRS-Leu(919) revealed yeast LeuRSs have a relaxed tRNA recognition capacity. We also studied the mis-activation and editing of non-cognate amino acids by CaLeuRS. Interestingly, we found that CaLeuRS is naturally deficient in tRNA-dependent pre-transfer editing for non-cognate norvaline while displaying a weak tRNA-dependent pre-transfer editing capacity for non-cognate ?-amino butyric acid. We also demonstrated that post-transfer editing of CaLeuRS is not tRNA(Leu) species-specific. In addition, other eukaryotic but not archaeal or bacterial LeuRSs were found to recognize CatRNA(Ser)(CAG). Overall, we systematically studied the aminoacylation and editing properties of CaLeuRS and established a characteristic LeuRS model with naturally deficient tRNA-dependent pre-transfer editing, which increases LeuRS types with unique editing patterns.
Project description:Leucyl-tRNA (transfer RNA) synthetase (LeuRS) is a multi-domain enzyme, which is divided into bacterial and archaeal/eukaryotic types. In general, one specific LeuRS, the domains of which are of the same type, exists in a single cell compartment. However, some species, such as the haloalkaliphile Natrialba magadii, encode two cytoplasmic LeuRSs, NmLeuRS1 and NmLeuRS2, which are the first examples of naturally occurring chimeric enzymes with different domains of bacterial and archaeal types. Furthermore, N. magadii encodes typical archaeal tRNA(Leu)s. The tRNA recognition mode, aminoacylation and translational quality control activities of these two LeuRSs are interesting questions to be addressed. Herein, active NmLeuRS1 and NmLeuRS2 were successfully purified after gene expression in Escherichia coli. Under the optimized aminoacylation conditions, we discovered that they distinguished cognate NmtRNA(Leu) in the archaeal mode, whereas the N-terminal region was of the bacterial type. However, NmLeuRS1 exhibited much higher aminoacylation and editing activity than NmLeuRS2, suggesting that NmLeuRS1 is more likely to generate Leu-tRNA(Leu) for protein biosynthesis. Moreover, using NmLeuRS1 as a model, we demonstrated misactivation of several non-cognate amino acids, and accuracy of protein synthesis was maintained mainly via post-transfer editing. This comprehensive study of the NmLeuRS/tRNA(Leu) system provides a detailed understanding of the coevolution of aminoacyl-tRNA synthetases and tRNA.
Project description:The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.
Project description:Leucyl-tRNA synthetase (LeuRS) has an insertion domain, called connective peptide 2 (CP2), either directly preceding or following the editing domain (CP1 domain), depending on the species. The global structures of the CP2 domains from all LeuRSs are similar. Although the CP1 domain has been extensively explored to be responsible for hydrolysis of mischarged tRNALeu, the role of the CP2 domain remains undefined. In the present work, deletion of the CP2 domain of Giardia lamblia LeuRS (GlLeuRS) showed that the CP2 domain is indispensable for amino acid activation and post-transfer editing and that it contributes to LeuRS-tRNALeu binding affinity. In addition, its functions are conserved in both eukaryotic/archaeal and prokaryotic LeuRSs from G. lamblia, Pyrococcus horikoshii (PhLeuRS), and Escherichia coli (EcLeuRS). Alanine scanning and site-directed mutagenesis assays of the CP2 domain identified several residues that are crucial for its various functions. Data from the chimeric mutants, which replaced the CP2 domain of GlLeuRS with either PhLeuRS or EcLeuRS, showed that the CP2 domain of PhLeuRS but not that of EcLeuRS can partially restore amino acid activation and post-transfer editing functions, suggesting that the functions of the CP2 domain are dependent on its location in the primary sequence of LeuRS.
Project description:aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNA(Leu). The editing domain of LeuRS deacylates the mischarged Ile-tRNA(Leu) and Met-tRNA(Leu). We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 A, 2.4 A, and 3.2 A resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.
Project description:To prevent genetic code ambiguity due to misincorporation of amino acids into proteins, aminoacyl-tRNA synthetases have evolved editing activities to eliminate intermediate or final non-cognate products. In this work we studied the different editing pathways of class Ia leucyl-tRNA synthetase (LeuRS). Different mutations and experimental conditions were used to decipher the editing mechanism, including the recently developed compound AN2690 that targets the post-transfer editing site of LeuRS. The study emphasizes the crucial importance of tRNA for the pre- and post-transfer editing catalysis. Both reactions have comparable efficiencies in prokaryotic Aquifex aeolicus and Escherichia coli LeuRSs, although the E. coli enzyme favors post-transfer editing, whereas the A. aeolicus enzyme favors pre-transfer editing. Our results also indicate that the entry of the CCA-acceptor end of tRNA in the editing domain is strictly required for tRNA-dependent pre-transfer editing. Surprisingly, this editing reaction was resistant to AN2690, which inactivates the enzyme by forming a covalent adduct with tRNA(Leu) in the post-transfer editing site. Taken together, these data suggest that the binding of tRNA in the post-transfer editing conformation confers to the enzyme the capacity for pre-transfer editing catalysis, regardless of its capacity to catalyze post-transfer editing.
Project description:Leucyl-tRNA synthetases (LeuRSs) catalyze the linkage of leucine with tRNA(Leu). LeuRS contains a catalysis domain (aminoacylation) and a CP1 domain (editing). CP1 is inserted 35 Å from the aminoacylation domain. Aminoacylation and editing require CP1 to swing to the coordinated conformation. The neck between the CP1 domain and the aminoacylation domain is defined as the CP1 hairpin. The location of the CP1 hairpin suggests a crucial role in the CP1 swing and domain-domain interaction. Here, the CP1 hairpin of Homo sapiens cytoplasmic LeuRS (hcLeuRS) was deleted or substituted by those from other representative species. Lack of a CP1 hairpin led to complete loss of aminoacylation, amino acid activation, and tRNA binding; however, the mutants retained post-transfer editing. Only the CP1 hairpin from Saccharomyces cerevisiae LeuRS (ScLeuRS) could partly rescue the hcLeuRS functions. Further site-directed mutagenesis indicated that the flexibility of small residues and the charge of polar residues in the CP1 hairpin are crucial for the function of LeuRS.
Project description:Leucyl-tRNA synthetase (LeuRS) has been identified as a target for a novel class of boron-containing small molecules that bind to its editing active site. When the 3' end of tRNA(Leu) binds to the editing active site, the boron cross-links to the cis-diols of its terminal ribose. The cross-linked RNA-protein complex blocks the overall aminoacylation activity of the enzyme. Similar to those of other LeuRSs, the human cytoplasmic enzyme (hscLeuRS) editing active site resides in a discrete domain called the connective polypeptide 1 domain (CP1), where mischarged tRNA binds for hydrolysis of the noncognate amino acid. The editing site of hscLeuRS includes a highly conserved threonine discriminator and universally conserved aspartic acid that were mutationally characterized. Substitution of the threonine residue to alanine uncoupled specificity as in other LeuRSs. However, the introduction of bulky residues into the amino acid binding pocket failed to block deacylation of tRNA, indicating that the architecture of the amino acid binding pocket is different compared to that of other characterized LeuRSs. In addition, mutation of the universally conserved aspartic acid abolished tRNA(Leu) deacylation. Surprisingly though, this editing-defective hscLeuRS maintained fidelity. It is possible that an alternate editing mechanism may have been activated upon failure of the post-transfer editing active site.
Project description:A conserved structural module following the KMSKS catalytic loop exhibits ?-?-?-? topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third ?-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNA(Leu), which decreases both aminoacylation and editing activities. Two glycine residues on this ?-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the ?-strand enhance the editing activity of EcLeuRS and sense the size of the tRNA(Leu) D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS.
Project description:Human mitochondrial leucyl-tRNA synthetase (hs mt LeuRS) achieves high aminoacylation fidelity without a functional editing active site, representing a rare example of a class I aminoacyl-tRNA synthetase (aaRS) that does not proofread its products. Previous studies demonstrated that the enzyme achieves high selectivity by using a more specific synthetic active site that is not prone to errors under physiological conditions. Interestingly, the synthetic active site of hs mt LeuRS displays a high degree of homology with prokaryotic, lower eukaryotic, and other mitochondrial LeuRSs that are less specific. However, there is one residue that differs between hs mt and Escherichia coli LeuRSs located on a flexible closing loop near the signature KMSKS motif. Here we describe studies indicating that this particular residue (K600 in hs mt LeuRS and L570 in E. coli LeuRS) strongly impacts aminoacylation in two ways: it affects both amino acid discrimination and transfer RNA (tRNA) binding. While this residue may not be in direct contact with the amino acid or tRNA substrate, substitutions of this position in both enzymes lead to altered catalytic efficiency and perturbations to the discrimination of leucine and isoleucine. In addition, tRNA recognition and aminoacylation is affected. These findings indicate that the conformation of the synthetic active site, modulated by this residue, may be coupled to specificity and provide new insights into the origins of selectivity without editing.