Structural snapshots of the KMSKS loop rearrangement for amino acid activation by bacterial tyrosyl-tRNA synthetase

Tyrosyl-tRNA synthetase (TyrRS) has been studied extensively by mutational and structural analyses to elucidate its catalytic mechanism. TyrRS has the HIGH and KMSKS motifs that catalyze the amino acid activation with ATP. In the present study, the crystal structures of the Escherichia coli TyrRS catalytic domain, in complexes with l-tyrosine and a l-tyrosyladenylate analogue, Tyr-AMS, were solved at 2.0A and 2.7A resolution, respectively. In the Tyr-AMS-bound structure, the 2'-OH group and adenine ring of the Tyr-AMS are strictly recognized by hydrogen bonds. This manner of hydrogen-bond recognition is conserved among the class I synthetases. Moreover, a comparison between the two structures revealed that the KMSKS loop is rearranged in response to adenine moiety binding and hydrogen-bond formation, and the KMSKS loop adopts the more compact ("semi-open") form, rather than the flexible, open form. The HIGH motif initially recognizes the gamma-phosphate, and then the alpha and gamma-phosphates of ATP, with a slight rearrangement of the residues. The other residues around the substrate also accommodate the Tyr-AMS. This induced-fit form presents a novel "snapshot" of the amino acid activation step in the aminoacylation reaction by TyrRS. The present structures and the T.thermophilus TyrRS ATP-free and bound structures revealed that the extensive induced-fit conformational changes of the KMSKS loop and the local conformational changes within the substrate binding site form the basis for driving the amino acid activation step: the KMSKS loop adopts the open form, transiently shifts to the semi-open conformation according to the adenosyl moiety binding, and finally assumes the rigid ATP-bound, closed form. After the amino acid activation, the KMSKS loop adopts the semi-open form again to accept the CCA end of tRNA for the aminoacyl transfer reaction.     

Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Class I aminoacyl-tRNA synthetases (aaRSs) use a Rossmann-fold domain to catalyze the synthesis of aminoacyl-tRNAs required for decoding genetic information. While the Rossmann-fold domain is conserved in evolution, the acceptor stem near the aminoacylation site varies among tRNA substrates, raising the question of how the conserved protein fold adapts to RNA sequence variations. Of interest is the existence of an unpaired C-A mismatch at the 1-72 position unique to bacterial initiator tRNA(fMet) and absent from elongator tRNAs. Here we show that the class I methionyl-tRNA synthetase (MetRS) of Escherichia coli and its close structural homolog cysteinyl-tRNA synthetase (CysRS) display distinct patterns of recognition of the 1-72 base pair. While the structural homology of the two enzymes in the Rossmann-fold domain is manifested in a common burst feature of aminoacylation kinetics, CysRS discriminates against unpaired 1-72, whereas MetRS lacks such discrimination. A structure-based alignment of the Rossmann fold identifies the insertion of an α-helical motif, specific to CysRS but absent from MetRS, which docks on 1-72 and may discriminate against mismatches. Indeed, substitutions of the CysRS helical motif abolish the discrimination against unpaired 1-72. Additional structural alignments reveal that with the exception of MetRS, class I tRNA synthetases contain a structural motif that docks on 1-72. This work demonstrates that by flexible insertion of a structural motif to dock on 1-72, the catalytic domain of class I tRNA synthetases can acquire structural plasticity to adapt to changes at the end of the tRNA acceptor stem.     

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.     

Amino acid activation of a dual-specificity tRNA synthetase is independent of tRNA

Transfer RNA can play a role in amino acid activation by aminoacyl-tRNA synthetases. For the prolyl-tRNA synthetase (ProRS) of Methanococcus jannaschii, which activates both proline and cysteine, the role of tRNA in amino acid selection and activation is of interest in the effort to understand the mechanism of the dual-specificity. While activation of proline does not require tRNA, whether or not tRNA is required in the activation of cysteine has been a matter of debate. Here, investigation of a series of buffer conditions shows that activation of cysteine occurs without tRNA in a wide-range of buffers. However, the extent of cysteine activation is strongly buffer-dependent, varying over a 180-fold range. In contrast, the extent of proline activation is much less sensitive to buffer conditions, varying over only a 36-fold range. We also find that addition of tRNA has a small threefold stimulatory effect on cysteine activation. The lack of a major role of tRNA in activation of cysteine suggests that the dual-specificity enzyme must distinguish cysteine from proline directly, without the assistance of each cognate tRNA, to achieve the necessary specificity required for protein synthesis.     

Crystal structure of leucyl-tRNA synthetase from the archaeon Pyrococcus horikoshii reveals a novel editing domain orientation

The editing domains of the closely homologous leucyl, isoleucyl, and valyl-tRNA synthetases (LeuRS, IleRS, and ValRS, respectively) contribute to accurate aminoacylation, by hydrolyzing misformed non-cognate aminoacyl-tRNAs. The editing domain is inserted at the same point of the sequence in IleRS, ValRS, and the archaeal/eukaryal LeuRS, but at a distinct point in the bacterial LeuRS. Here, we showed that LeuRS from the archaeon Pyrococcus horikoshii has editing activity against the nearly cognate isoleucine. The conserved Asp332 in the editing domain is crucial for this activity. A deletion mutant lacking the C-terminal region has only negligible aminoacylation activity, but retains the full activity of adenylate synthesis and editing. We determined the crystal structure of this editing-active, truncated form of P.horikoshii LeuRS at 2.1 A resolution. The structure revealed that it has a novel editing domain orientation. The editing domain of P.horikoshii LeuRS is rotated by approximately 180 degrees (rotational state II), with the two-beta-stranded linker untwisted by a half-turn, as compared to those in IleRS and ValRS (rotational state I). This editing domain rotational state in the archaeal LeuRS is similar to that in the bacterial LeuRS. However, because of the insertion point difference, the orientation of the editing domain relative to the enzyme core in the archaeal LeuRS differs completely from that in the bacterial LeuRS. An insertion region specific to the archaeal/eukaryal LeuRS editing domains interacts with the enzyme core and stabilizes the unique orientation. Thus, we established that there are three types of editing domain orientations relative to the enzyme core, depending on the combination of the editing domain insertion point (i or ii) and the rotational state (I or II): [i, I] for IleRS and ValRS, [ii, II] for the bacterial LeuRS, and now [i, II] for the archaeal/eukaryal LeuRS.     

Concurrent molecular recognition of the amino acid and tRNA by a ribozyme.

We have recently reported an in vitro-evolved precursor tRNA (pre-tRNA) that is able to catalyze aminoacylation on its own 3'-hydroxyl group. This catalytic pre-tRNA is susceptible to RNase P RNA, generating the 5'-leader ribozyme and mature tRNA. The 5'-leader ribozyme is also capable of aminoacylating the tRNA in trans, thus acting as an aminoacyl-tRNA synthetase-like ribozyme (ARS-like ribozyme). Here we report its structural characterization that reveals the essential catalytic core. The ribozyme consists of three stem-loops connected by two junction regions. The chemical probing analyses show that a U-rich region (U59-U62 in J2a/3 and U67-U68 in L3) of the ribozyme is responsible for the recognition of the phenylalanine substrate. Moreover, a GGU-motif (G70-U72) of the ribozyme, adjacent to the U-rich region, forms base pairs with the tRNA 3' terminus. Our demonstration shows that simple RNA motifs can recognize both the amino acid and tRNA simultaneously, thus aminoacylating the 3' terminus of tRNA in trans.     

A sulfur amino acid deficiency changes the amino acid composition of body protein in piglets

Experiments carried out to determine the amino acid requirement in growing animals are often based on the premise that the amino acid composition of body protein is constant. However, there are indications that this assumption may not be correct. The objective of this study was to test the effect of feeding piglets a diet deficient or not in total sulfur amino acids (TSAA; Met + Cys) on nitrogen retention and amino acid composition of proteins in different body compartments. Six blocks of three pigs each were used in a combined comparative slaughter and nitrogen balance study. One piglet in each block was slaughtered at 42 days of age, whereas the other piglets received a diet deficient or not in TSAA for 19 days and were slaughtered thereafter. Two diets were formulated to provide either 0.20% Met and 0.45% TSAA (on a standardized ileal digestible basis) or 0.46% Met and 0.70% TSAA. Diets were offered approximately 25% below ad libitum intake. At slaughter, the whole animal was divided into carcass, blood, intestines, liver, and the combined head, tail, feet and other organs (HFTO), which were analyzed for nitrogen and amino acid contents. Samples of the longissimus muscle (LM) were analyzed for myosin heavy chain (MyHC) and actin contents. Nitrogen retention was 20% lower in piglets receiving the TSAA-deficient diet (P < 0.01). In these piglets, the nitrogen content in tissue gain was lower in the empty body, carcass, LM and blood (P < 0.05) or tended to be lower in HFTO (P < 0.10), but was not different in the intestines and liver. The Met content in retained protein was lower in the empty body, LM and blood (P < 0.05), and tended to be lower in the carcass (P < 0.10). The Cys content was lower in LM, but higher in blood of piglets receiving the TSAA-deficient diet (P < 0.05). Skeletal muscle appeared to be affected most by the TSAA deficiency. In LM, the Met content in retained protein was reduced by 12% and total Met retention by more than 60%. The MyHC and actin contents in LM were not affected by the TSAA content of the diet. These results show that a deficient TSAA supply affects the amino acid composition of different body proteins. This questions the use of a constant ideal amino acid profile to express dietary amino acid requirements, but also illustrates the plasticity of the animal to cope with nutritional challenges.     

Inhibition of Arabidopsis growth by the allelopathic compound azetidine‐2‐carboxylate is due to the low amino acid specificity of cytosolic prolyl‐tRNA synthetase

The toxicity of azetidine‐2‐carboxylic acid (A2C), a structural analogue of L‐proline, results from its incorporation into proteins due to misrecognition by prolyl‐tRNA synthetase (ProRS). The growth of Arabidopsis thaliana seedling roots is more sensitive to inhibition by A2C than is cotyledon growth. Arabidopsis contains two ProRS isozymes. AtProRS‐Org (At5g52520) is localized in chloroplasts/mitochondria, and AtProRS‐Cyt (At3g62120) is cytosolic. AtProRS‐Cyt mRNA is more highly expressed in roots than in cotyledons. Arabidopsis ProRS isoforms were expressed as His‐tagged recombinant proteins in Escherichia coli. Both enzymes were functionally active in ATP‐PPi exchange and aminoacylation assays, and showed similar K_m for L‐proline. A major difference was observed in the substrate specificity of the two enzymes. AtProRS‐Cyt showed nearly identical substrate specificity for L‐proline and A2C, but for AtProRS‐Org the specificity constant was 77.6 times higher for L‐proline than A2C, suggesting that A2C‐sensitivity may result from lower amino acid specificity of AtProRS‐Cyt. Molecular modelling and simulation results indicate that this specificity difference between the AtProRS isoforms may result from altered modes of substrate binding. Similar kinetic results were obtained with the ProRSs from Zea mays, suggesting that the difference in substrate specificity is a conserved feature of ProRS isoforms from plants that do not accumulate A2C and are sensitive to A2C toxicity. The discovery of the mode of action of A2C toxicity could lead to development of biorational weed management strategies.     

Differential coupling efficiency of chemically activated amino acid to tRNA

Summary Interaction based on possible chemical affinity of an amino acid for tRNA was examined as a model for the aminoacylation of primitive tRNA without aid of an enzyme system. Two types of reaction were carried out and compared. One was the acyl linkage of amino acid to the 5-terminal phosphate of a tRNA activated as an imidazolide. The other was the incorporation of an amino acid activated as an imidazolide into 2(3)-hydroxyl groups of intact tRNA. Both types of reaction indicated that none of the amino acids tested had any selectivity for the tRNAs examined. However, the rates of reaction with a given tRNA were different among amino acids. In the second type of reaction, amino acids were found mainly at loop-out regions of tRNA, but not at either its 5- or 3-terminal sitesOneA ₂₆₀ unit is defined as an amount of material which gives an absorption of 1.0 at 260 nm when dissolved in 1 ml water and measured with a 1-cm light path     

Peripheral neuropathy via mutant tRNA synthetases: Inhibition of protein translation provides a possible explanation

Recent evidence indicates that inhibition of protein translation may be a common pathogenic mechanism for peripheral neuropathy associated with mutant tRNA synthetases (aaRSs). aaRSs are enzymes that ligate amino acids to their cognate tRNA, thus catalyzing the first step of translation. Dominant mutations in five distinct aaRSs cause Charcot‐Marie‐Tooth (CMT) peripheral neuropathy, characterized by length‐dependent degeneration of peripheral motor and sensory axons. Surprisingly, loss of aminoacylation activity is not required for mutant aaRSs to cause CMT. Rather, at least for some mutations, a toxic‐gain‐of‐function mechanism underlies CMT‐aaRS. Interestingly, several mutations in two distinct aaRSs were recently shown to inhibit global protein translation in Drosophila models of CMT‐aaRS, by a mechanism independent of aminoacylation, suggesting inhibition of translation as a common pathogenic mechanism. Future research aimed at elucidating the molecular mechanisms underlying the translation defect induced by CMT‐mutant aaRSs should provide novel insight into the molecular pathogenesis of these incurable diseases.     

Thermostabilization of porcine kidney D-amino acid oxidase by a single amino acid substitution

D-amino acid oxidase (DAO) is of considerable practical importance, such as bioconversion and enzymatic assay. In this study, we succeeded in obtaining a thermostable mutant DAO from porcine kidney by a single amino acid substitution. This mutant enzyme, F42C, was stable at 55 degrees C, while the wild-type enzyme was stable only up to 45 degrees C. The Km values of F42C for D-amino acids was about half of those of the wild-type enzyme. This mutant DAO with improved stability and affinity for its substrates is advantageous for the determination of D-amino acids.     

Editing by a tRNA synthetase: DNA aptamer-induced translocation and hydrolysis of a misactivated amino acid

Aminoacyl-tRNA synthetases establish the rules of the genetic code by aminoacylation reactions. Occasional activation of the wrong amino acid can lead to errors of protein synthesis. For isoleucyl-tRNA synthetase, these errors are reduced by tRNA-dependent hydrolytic editing reactions that occur at a site 25 A from the active site. These reactions require that the misactivated amino acid be translocated from the active site to the center for editing. One mechanism describes translocation as requiring the mischarging of tRNA followed by a conformational change in the tRNA that moves the amino acid from one site to the other. Here a specific DNA aptamer is investigated. The aptamer can stimulate amino acid-specific editing but cannot be aminoacylated. Although the aptamer could in principle stimulate hydrolysis of a misactivated amino acid by an idiosyncratic mechanism, the aptamer is shown here to induce translocation and hydrolysis of misactivated aminoacyl adenylate at the same site as that seen with the tRNA cofactor. Thus, translocation to the site for editing does not require joining of the amino acid to the nucleic acid. Further experiments demonstrated that aptamer-induced editing is sensitive to aptamer sequence and that the aptamer is directed to a site other than the active site or tRNA binding site of the enzyme.     

Predicting the functional consequences of non-synonymous single nucleotide polymorphisms: structure-based assessment of amino acid variation

We have developed a formalism and a computational method for analyzing the potential functional consequences of non-synonymous single nucleotide polymorphisms. Our approach uses a structural model and phylogenetic information to derive a selection of structure and sequence-based features serving as indicators of an amino acid polymorphim's effect on function. The feature values can be integrated into a probabilistic assessment of whether an amino acid polymorphism will affect the function or stability of a target protein. The method has been validated with data sets of unbiased mutations in the lac repressor and lysoyzyme. Applying our methodology to recent surveys of genetic variation in the coding regions of clinically important genes, we estimate that approximately 26-32 % of the natural non-synonymous single nucleotide polymorphisms have effects on function. This estimate suggests that a typical person will have about 6240-12,800 heterozygous loci that encode proteins with functional variation due to natural amino acid polymorphism.     

Improvement of Yersinia frederiksenii phytase performance by a single amino acid substitution

A new phytase (APPA) with optimum pH 2.5—substantially lower than that of most of microbial phytases (pH 4.5–6.0)—was cloned from Yersinia frederiksenii and heterologously expressed in Escherichia coli. Containing the highly conserved motifs typical of histidine acid phosphatases, APPA has the highest identity (84%) to the Yersinia intermedia phytase (optimal pH 4.5), a member of histidine acid phosphatase family. Based on sequence alignment and molecular modeling of APPA and related phytases, APPA has only one divergent residue, Ser51, in close proximity to the catalytic site. To understand the acidic adaptation of APPA, five mutants (S51A, S51T, S51D, S51K, and S51I) were constructed by site‐directed mutagenesis, expressed in E. coli, purified, and characterized. Mutants S51T and S51I exhibited a shift in the optimal pH from 2.5 to 4.5 and 5.0, respectively, confirming the role of Ser51 in defining the optimal pH. Thus, a previously unrecognized factor other than electrostatics—presumably the side‐chain structure near the active site—contributes to the optimal pH for APPA activity. Compared with wild‐type APPA, mutant S51T showed higher specific activity, greater activity over pH 2.0–5.5, and increased thermal and acid stability. These properties make S51T a better candidate than the wild‐type APPA for use in animal feed. Biotechnol. Bioeng. 2009;103: 857–864. © 2009 Wiley Periodicals, Inc.     

Predicting disease-associated substitution of a single amino acid by analyzing residue interactions

Background  

The rapid accumulation of data on non-synonymous single nucleotide polymorphisms (nsSNPs, also called SAPs) should allow us to further our understanding of the underlying disease-associated mechanisms. Here, we use complex networks to study the role of an amino acid in both local and global structures and determine the extent to which disease-associated and polymorphic SAPs differ in terms of their interactions to other residues.     

Extension of the COG and arCOG databases by amino acid and nucleotide sequences

Background  

The current versions of the COG and arCOG databases, both excellent frameworks for studies in comparative and functional genomics, do not contain the nucleotide sequences corresponding to their protein or protein domain entries.     

Specificity of elongation factor Tu from Escherichia coli with respect to attachment to the amino acid to the 2' or 3'-hydroxyl group of the terminal adenosine of tRNA.

Modified Tyr-tRNATyr and Phe-tRNAPhe species from yeast having the aminoacyl residue bound specifically to the 2' and 3' position of the terminal adenosine, respectively, were investigated for their ability to form ternary complexes with Escherichia coli elongation factor Tu and GTP. Both Tyr-tRNATyr-CpCpA (2'd) and Tyr-tRNATyr-CpCpA(3' d) derivatives which are esterified with the amino acid on the 3' and 2' position respectively and which lack the vicinal hydroxyl were able to form ternary complexes. The stability of these ternary complexes was lower than in the case of native Tyr-tRNATyr-CpCpA. Tyr-tRNATyr-CpCpA(3' d) having the amino acid attached to the 2' position interacted considerably more strongly with EF-Tu - GTP than Tyr-tRNATyr-CpCpA(2' d). Ternary complex formation was observed with neither Phe-tRNAPhe-CpCpA(2'NH2) nor Phe-tRNAPhe-CpCpA(3'NH2). It is concluded that 2' as well as 3' isomers of native aminoacyl-tRNA can be utilized for ternary complex formation but in a following step a uniform 2'-aminoacyl-tRNA - EF-Tu - GTP complex is formed. Although the free vicinal hydroxyl group of the terminal adenosine is not absolutely required, replacement of the ester linkage through with the amino acid is attached to tRNA by an amide linkage leads to loss of ability to interact with elongation factor Tu.     

FGFR3 dimer stabilization due to a single amino acid pathogenic mutation

Mutations in the transmembrane (TM) domains of receptor tyrosine kinases (RTKs) have been implicated in the induction of pathological phenotypes. These mutations are believed to stabilize the RTK dimers, and thus promote unregulated signaling. However, the energetics behind the pathology induction has not been determined. An example of a TM domain pathogenic mutation is the Ala391-->Glu mutation in fibroblast growth factor receptor 3 (FGFR3), linked to Crouzon syndrome with acanthosis nigricans, as well as to bladder cancer. Here, we determine the free energy of dimerization of wild-type and mutant FGFR3 TM domain in lipid bilayers using F?rster resonance energy transfer, and we show that hydrogen bonding between Glu391 and the adjacent helix in the dimer is a feasible mechanism for dimer stabilization. The measured change in the free energy of dimerization due to the Ala391-->Glu pathogenic mutation is -1.3 kcal/mol, consistent with previous reports of hydrogen bond strengths in proteins. This is the first quantitative measurement of mutant RTK stabilization in a membrane environment. We show that this seemingly modest value can lead to a large increase in dimer fraction and thus profoundly affect RTK-mediated signal transduction.