CP1 Domain Research Articles

Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase.

The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 A. Crystals diffract to beyond 1.8 A resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 A resolution. All structures were solved by molecular-replacement techniques.

Leucyl-tRNA synthetase from the ancestral bacterium Aquifex aeolicus contains relics of synthetase evolution

The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (alphabeta-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNA(Leu) and minihelix(Leu). Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplanted into the inactive Escherichia coli LeuRS editing domain. Likewise, fusion of the beta-subunit of alphabeta-LeuRS to the E. coli editing domain activates its editing function. These results suggest that alphabeta-LeuRS still carries the basic features from a primitive synthetase molecule. It has a remarkable capacity to transfer autonomous active modules, which is consistent with the idea that modern synthetases arose after exchange of small idiosyncratic domains. It also has a unique alphabeta-heterodimeric structure with separated catalytic and tRNA-binding sites. Such an organization supports the tRNA/synthetase coevolution theory that predicts sequential addition of tRNA and synthetase domains.

Crystallization and preliminary X-ray crystallographic study of the editing domain of Thermus thermophilus isoleucyl-tRNA synthetase complexed with pre- and post-transfer editing-substrate analogues.

The CP1 domain (the editing domain) of isoleucyl-tRNA synthetase (IleRS) hydrolyzes misactivated Val-AMP in pre-transfer editing and mischarged Val-tRNA(Ile) in post-transfer editing. The CP1 domain of Thermus thermophilus IleRS was expressed in isolation, purified and cocrystallized with Val-AMS (a Val-AMP analogue) and with Val-2AA (a Val-tRNA(Ile) analogue). Two different expression constructs were used for each cocrystallization. The complex crystals with Val-AMS belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 102.00, c = 84.88 A. The asymmetric unit contains two molecules of the CP1 domain, with a corresponding crystal volume per protein weight of 2.7 A3 Da(-1) and a solvent content of 53.5%. The complex crystals with Val-2AA belong to the tetragonal space group P4(1)22, with unit-cell parameters a = b = 72.59, c = 83.68 A. The asymmetric unit contains one molecule of the CP1 domain, with a corresponding crystal volume per protein weight of 2.8 A3 Da(-1) and a solvent content of 55.8%. Data sets diffracting to 1.7 A resolution were collected from each single crystal at 100 K.

Groups on the side chain of T252 in Escherichia coli leucyl-tRNA synthetase are important for discrimination of amino acids and cell viability

Leucyl-tRNA synthetase (LeuRS) catalyzes the leucylation of tRNA Leu. To maintain the fidelity of protein biosynthesis, LeuRS also catalyzes the editing reaction. In the present work, highly conserved T252 in the T-rich region within CP1 domain of Escherichia coli LeuRS was mutated to G, D, or E. Steady-state kinetic of aminoacylation, and combined editing assays indicated that not only the size of the amino acid but also the absence of hydrogen bonds between T252 and adjacent molecules may affect the editing. It is further confirmed by in vivo experiments using the temperature-sensitive strain KL231 (Δ leuS), which revealed the arrested growth of bacterial cells bearing mutants with highly impaired editing activity in the presence of leucine analog.

Crystal Structures of the CP1 Domain from Thermus thermophilus Isoleucyl-tRNA Synthetase and Its Complex with l-Valine

Isoleucyl-tRNA synthetase (IleRS) links tRNA(Ile) with not only its cognate isoleucine but also the nearly cognate valine. The CP1 domain of IleRS deacylates, or edits, the mischarged Val-tRNA(Ile). We determined the crystal structures of the Thermus thermophilus IleRS CP1 domain alone, and in its complex with valine at 1.8- and 2.0-A resolutions, respectively. In the complex structure, the Asp(328) residue, which was shown to be critical for the editing reaction against Val-tRNA(Ile) by a previous mutational analysis, recognizes the valine NH(3)(+) group. The valine side chain binding pocket is only large enough to accommodate valine, and the placement of an isoleucine model in this location revealed that the additional methylene group of isoleucine would clash with His(319). The H319A mutant of Escherichia coli IleRS reportedly deacylates the cognate Ile-tRNA(Ile) in addition to Val-tRNA(Ile), indicating that the valine-binding mode found in this study represents that in the Val-tRNA(Ile) editing reaction. Analyses of the Val-tRNA(Ile) editing activities of T. thermophilus IleRS mutants revealed the importance of Thr(228), Thr(229), Thr(230), and Asp(328), which are coordinated with water molecules in the present structure. The structural model for the Val-adenosine moiety of Val-tRNA(Ile) bound in the IleRS editing site revealed some interesting differences in the substrate binding and recognizing mechanisms between IleRS and T. thermophilus leucyl-tRNA synthetase. For example, the carbonyl oxygens of the amino acids are located opposite to each other, relative to the adenosine ribose ring, and are differently recognized.

Elucidation of tRNA-dependent editing by a class II tRNA synthetase and significance for cell viability.

Editing of misactivated amino acids by class I tRNA synthetases is encoded by a specialized internal domain specific to class I enzymes. In contrast, little is known about editing activities of the structurally distinct class II enzymes. Here we show that the class II alanyl-tRNA synthetase (AlaRS) has a specialized internal domain that appears weakly related to an appended domain of threonyl-tRNA synthetase (ThrRS), but is unrelated to that found in class I enzymes. Editing of misactivated glycine or serine was shown to require a tRNA cofactor. Specific mutations in the aforementioned domain disrupt editing and lead to production of mischarged tRNA. This class-specific editing domain was found to be essential for cell growth, in the presence of elevated concentrations of glycine or serine. In contrast to ThrRS, where the editing domain is not found in all three kingdoms of living organisms, it was incorporated early into AlaRSs and is present throughout evolution. Thus, tRNA-dependent editing by AlaRS may have been critical for making the genetic code sufficiently accurate to generate the tree of life.

An inserted region of leucyl-tRNA synthetase plays a critical role in group I intron splicing.

Yeast mitochondrial leucyl-tRNA synthetase (LeuRS) binds to the bI4 intron and collaborates with the bI4 maturase to aid excision of the group I intron. Deletion analysis isolated the inserted LeuRS CP1 domain as a critical factor in the protein's splicing activity. Protein fragments comprised of just the LeuRS CP1 region rescued complementation of a yeast strain that expressed a splicing-defective LeuRS. Three-hybrid analysis determined that these CP1-containing LeuRS fragments, ranging from 214 to 375 amino acids, bound to the bI4 intron. In each case, interactions with only the LeuRS protein fragment specifically stimulated bI4 intron splicing activity. Substitution of a homologous CP1 domain from isoleucyl-tRNA synthetase or mutation within the LeuRS CP1 region of the smallest protein fragment abolished RNA binding and splicing activity. The CP1 domain is best known for its amino acid editing activity. However, these results suggest that elements within the LeuRS CP1 domain also play a novel role, independent of the full-length tRNA synthetase, in binding the bI4 group I intron and facilitating its self-splicing activity.

A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu.

Aminoacyl-tRNA synthetases ensure the fidelity of protein synthesis by accurately selecting and activating cognate amino acids for aminoacylation of the correct tRNA. Some tRNA synthetases have evolved an editing active site that is separate from the amino acid activation site providing two steps or "sieves" for amino acid selection. These two sieves rely on different strategies for amino acid recognition to significantly enhance the accuracy of aminoacylation. We have performed alanine scanning mutagenesis in a conserved threonine-rich region of the Escherichia coli leucyl-tRNA synthetase's CP1 domain that is hypothesized to contain a putative editing active site. Characterization of purified mutant proteins led to the identification of a single conserved threonine that prevents the cognate leucine amino acid from being hydrolyzed after aminoacylation of the tRNA. Mutation of this threonine to an alanine eliminates discrimination of the cognate amino acid in the editing active site. This provides a molecular example of an amino acid discrimination mechanism in the tRNA synthetase's editing active site.

CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function.

The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve", while the reaction products of amino acids smaller than the cognate substrate will go through a "fine sieve" and be hydrolyzed. This "double-sieve" mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain). This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNA(Leu) by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA(Leu) made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function.

The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity.

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.