The 2.0 A crystal structure of Thermus thermophilus methionyl-tRNA synthetase reveals two RNA-binding modules

Background: The 20 aminoacyl-tRNA synthetases are divided into two classes, I and II. The 10 class I synthetases are considered to have in common the catalytic domain structure based on the Rossmann fold, which is totally different from the class II catalytic domain structure. The class I synthetases are further divided into three subclasses, a, b and c, according to sequence homology. No conserved structural features for tRNA recognition by class I synthetases have been established. Results: We determined the crystal structure of the class Ia methionyl-tRNA synthetase (MetRS) at 2.0 A resolution, using MetRS from an extreme thermophile, Thermus thermophilus HB8. The T. thermophilus MetRS structure is in full agreement with the biochemical and genetic data from Escherichia coli MetRS. The conserved 'anticodon-binding' residues are spatially clustered on an alpha-helix-bundle domain. The Rossmann-fold and anticodon-binding domains are connected by a beta-alpha-alpha-beta-alpha topology ('SC fold') domain that contains the class I specific KMSKS motif. Conclusions: The alpha-helix-bundle domain identified in the MetRS structure is the signature of the class Ia enzymes, as it was also identified in the class Ia structures of the isoleucyl- and arginyl-tRNA synthetases. The beta-alpha-alpha-beta-alpha topology domain, which can now be identified in all known structures of the class Ia and Ib synthetases, is likely to dock with the inner side of the L-shaped tRNA, thereby positioning the anticodon stem.     

Correlating amino acid conservation with function in tyrosyl-tRNA synthetase

Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). Previous investigations indicate that the class I conserved KMSKS motif is involved in only the first step of the reaction (i.e. tyrosine activation). Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step. In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction. These results imply that stabilization of the transition state for the first step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 3' end of tRNA. We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP.     

The crystal structure of E. coli pantothenate synthetase confirms it as a member of the cytidylyltransferase superfamily

BACKGROUND: Pantothenate synthetase (EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis. It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction. RESULTS: We describe the overexpression, purification, and crystal structure of recombinant pantothenate synthetase from E. coli. The structure was solved by a selenomethionine multiwavelength anomalous dispersion experiment and refined against native data to a final R(cryst) of 22.6% (R(free) = 24.9%) at 1.7 A resolution. The enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid. CONCLUSIONS: The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily. This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex.     

Sequence, linkage to H2-K, and function of mouse tapasin in MHC class I assembly

 Assembly of major histocompatibility complex (MHC) class I molecules in human cells is dependent on the accessory protein tapasin, which mediates their interaction with the transporters associated with antigen processing (TAP) and thereby ensures efficient peptide binding. Analysis of a mouse tapasin complementary DNA defined a conserved polypeptide sharing sequences diagnostic of a transmembrane protein related to the immunoglobulin superfamily, and an endoplasmic reticulum retention motif. The mouse tapasin gene was mapped about 70 kilobases from H2-K at the centromeric end of the mouse MHC. Expression of mouse tapasin in a tapasin-deficient human mutant cell line restored the normal assembly and expression of class I alleles. Thus, tapasin is a structurally and functionally conserved component of the MHC class I antigen processing pathway. Its genetic linkage to the class I and TAP subunit genes in the MHC may be of significance in the coordinate expression and functional coadaptation of the diverse gene products. Received: 1 February 1998 / Revised: 23 March 1998     

Role for a conserved structural motif in assembly of a class I aminoacyl-tRNA synthetase active site

The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzyme's cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.     

Functional role of the prokaryotic proline-tRNA synthetase insertion domain in amino acid editing

Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to cognate tRNAs in a two-step process that is critical for the faithful translation of genetic information. During the first chemical step of tRNA aminoacylation, noncognate amino acids that are smaller than or isosteric with the cognate substrate can be misactivated. Thus, to maintain high accuracy during protein translation, some synthetases have evolved an editing mechanism. Previously, we showed that class II Escherichia coli proline-tRNA synthetase (ProRS) is capable of (1) weakly misactivating Ala, (2) hydrolyzing the misactivated Ala-AMP in a reaction known as pretransfer editing, and (3) deacylating a mischarged Ala-tRNA(Pro) variant via a post-transfer editing pathway. In contrast to most systems where an editing function has been established, pretransfer editing by E. coli ProRS occurs in a tRNA-independent fashion. However, neither the pre- nor the post-transfer editing active site(s) has been identified. Sequence analyses revealed that most prokaryotic ProRSs possess a large insertion domain (INS) between class II conserved motifs 2 and 3. The function of the approximately 180-amino acid INS in E. coli ProRS is the subject of this investigation. Alignment-guided Ala scanning mutagenesis was carried out to test conserved amino acid residues present in the INS for their role in pre- and post-transfer editing. Our biochemical data and modeling studies suggest that the prokaryotic INS plays a critical role in editing and that this activity resides in a domain that is functionally and structurally distinct from the aminoacylation active site.     

The fidelity of the translation of the genetic code.

Aminoacyl-tRNA synthetases play a central role in maintaining accuracy during the translation of the genetic code. To achieve this challenging task they have to discriminate against amino acids that are very closely related not only in structure but also in chemical nature. A 'double-sieve' editing model was proposed in the late seventies to explain how two closely related amino acids may be discriminated. However, a clear understanding of this mechanism required structural information on synthetases that are faced with such a problem of amino acid discrimination. The first structural basis for the editing model came recently from the crystal structure of isoleucyl-tRNA synthetase, a class I synthetase, which has to discriminate against valine. The structure showed the presence of two catalytic sites in the same enzyme, one for activation, a coarse sieve which binds both isoleucine and valine, and another for editing, a fine sieve which binds only valine and rejects isoleucine. Another structure of the enzyme in complex with tRNA showed that the tRNA is responsible for the translocation of the misactivated amino-acid substrate from the catalytic site to the editing site. These studies were mainly focused on class I synthetases and the situation was not clear about how class II enzymes discriminate against similar amino acids. The recent structural and enzymatic studies on threonyl-tRNA synthetase, a class II enzyme, reveal how this challenging task is achieved by using a unique zinc ion in the active site as well as by employing a separate domain for specific editing activity. These studies led us to propose a model which emphasizes the mirror symmetrical approach of the two classes of enzymes and highlights that tRNA is the key player in the evolution of these class of enzymes.     

Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases

The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC )) generates the conserved CCA sequence responsible for the attachment of amino acid at the 3' terminus of tRNA molecules. It was shown that enzymes from various organisms strictly recognize the elbow region of tRNA formed by the conserved D- and T-loops. However, most of the mammalian mitochondrial (mt) tRNAs lack consensus sequences in both D- and T-loops. To characterize the mammalian mt CCA-adding enzymes, we have partially purified the enzyme from bovine liver mitochondria and determined cDNA sequences from human and mouse dbESTs by mass spectrometric analysis. The identified sequences contained typical amino-terminal peptides for mitochondrial protein import and had characteristics of the class II nucleotidyltransferase superfamily that includes eukaryotic and eubacterial CCA-adding enzymes. The human recombinant enzyme was overexpressed in Escherichia coli, and its CCA-adding activity was characterized using several mt tRNAs as substrates. The results clearly show that the human mt CCA-adding enzyme can efficiently repair mt tRNAs that are poor substrates for the E. coli enzyme although both enzymes work equally well on cytoplasmic tRNAs. This suggests that the mammalian mt enzymes have evolved so as to recognize mt tRNAs with unusual structures.     

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.     

Crystal structure of CTP:glycerol-3-phosphate cytidylyltransferase from Staphylococcus aureus: examination of structural basis for kinetic mechanism

Integrity of the cell wall is essential for bacterial survival, and as a consequence components involved in its biosynthesis can potentially be exploited as targets for antibiotics. One such potential target is CTP:glycerol-3-phosphate cytidylyltransferase. This enzyme (TarD(Sa) in Staphylococcus aureus and TagD(Bs) in Bacillus subtilis) catalyzes the formation of CDP-glycerol, which is used for the assembly of linkages between peptidoglycan and teichoic acid polymer in Gram-positive bacteria. Intriguingly, despite the high sequence identity between TarD(Sa) and TagD(Bs) (69% identity), kinetic studies show that these two enzymes differ markedly in their kinetic mechanism and activity. To examine the basis for the disparate enzymological properties, we have determined the crystal structure of TarD(Sa) in the apo state to 3 A resolution, and performed equilibrium sedimentation analysis. Comparison of the structure with that of CTP- and CDP-glycerol-bound TagD(Bs) crystal structures reveals that the overall structure of TarD(Sa) is essentially the same as that of TagD(Bs), except in the C-terminus, where it forms a helix in TagD(Bs) but is disordered in the apo TarD(Sa) structure. In addition, TarD(Sa) can exist both as a tetramer and as a dimer, unlike TagD(Bs), which is a dimer. These observations shed light on the structural basis for the differing kinetic characteristics between TarD(Sa) and TagD(Bs).     

Combinatorial analysis of loop nucleotides in human mitochondrial tRNALeu(UUR)

A series of disease-related mutations are known to affect the hs mt tRNA(Leu(UUR)) gene, and the molecular-level properties of this tRNA may underlie the effects of pathogenic sequence changes. A combinatorial approach has been used to explore the importance of the D, TPsiC, and anticodon loops of hs mt tRNA(Leu(UUR)) in the structure and function of this molecule. A tRNA library was constructed with 20 randomized nucleotides in the loop regions of hs mt tRNA(Leu(UUR)), and tRNA variants that were aminoacylated by hs mt LeuRS were isolated using an in vitro selection approach. Analysis of 26 selected sequences revealed that a stabilized anticodon stem significantly enhances aminoacylation activity. However, anticodon loop nucleotides were not conserved in the active sequences, indicating that this region of hs mt tRNA(Leu(UUR)) is not involved in recognition by LeuRS. Within the D and TPsiC loops, only two nucleotides conserved their identities, while new sequences were selected that likely mediate interloop interactions. The results indicate that hs mt tRNA(Leu(UUR)), which is known to have structurally weak D and anticodon stems, benefits functionally from the introduction of stabilizing interactions. However, the locations of individual nucleotides that govern discrimination of this tRNA by hs mt LeuRS still remain obscure.     

In silico detection of tRNA sequence features characteristic to aminoacyl-tRNA synthetase class membership

Aminoacyl tRNA synthetases (aaRS) are grouped into Class I and II based on primary and tertiary structure and enzyme properties suggesting two independent phylogenetic lineages. Analogously, tRNA molecules can also form two respective classes, based on the class membership of their corresponding aaRS. Although some aaRS-tRNA interactions are not extremely specific and require editing mechanisms to avoid misaminoacylation, most aaRS-tRNA interactions are rather stereospecific. Thus, class-specific aaRS features could be mirrored by class-specific tRNA features. However, previous investigations failed to detect conserved class-specific nucleotides. Here we introduce a discrete mathematical approach that evaluates not only class-specific 'strictly present', but also 'strictly absent' nucleotides. The disjoint subsets of these elements compose a unique partition, named extended consensus partition (ECP). By analyzing the ECP for both Class I and II tDNA sets from 50 (13 archaeal, 30 bacterial and 7 eukaryotic) species, we could demonstrate that class-specific tRNA sequence features do exist, although not in terms of strictly conserved nucleotides as it had previously been anticipated. This finding demonstrates that important information was hidden in tRNA sequences inaccessible for traditional statistical methods. The ECP analysis might contribute to the understanding of tRNA evolution and could enrich the sequence analysis tool repertoire.     

Comparative modeling of class 1 lysyl tRNA synthetase from Treponema pallidum

Lysyl tRNA synthetases facilitate amino acylation and play a crucial role in the essential cellular process of translation. They are grouped into two distinct classes (class I and class II). Class I lysyl tRNA synthetase is considered as a drug target for syphilis caused by Treponema pallidum. Comparative genome analysis shows the absence of its sequence homolog in eukaryotes. The structure of class I lysyl tRNA synthetase from Treponema pallidum is unknown and the difficulties in the in vitro culturing of Treponema makes it non-trivial. We used the structural template of class I lysyl tRNA synthetase from the archaea Pyrococcus horikoshii for modeling the Treponema pallidum lysyl tRNA synthetase structure. Thus, we propose the usefulness of the modeled class I lysyl tRNA synthetase for the design of suitable inhibitors towards the treatment of syphilis.     

Breaking the stereo barrier of amino acid attachment to tRNA by a single nucleotide

Aminoacyl-tRNA synthetases are responsible for attaching amino acid residues to the tRNA 3'-end. The two classes of synthetases approach tRNA as mirror images, with opposite but symmetrical stereochemistries that allow the class I enzymes to attach amino acid residues to the 2'-hydroxyl group of the terminal ribose, whereas, the class II enzymes attach amino acid residues to the 3'-hydroxyl group. However, we show here that the attachment of cysteine to tRNA(Cys) by the class I cysteinyl-tRNA synthetase (CysRS) is flexible; the enzyme is capable of using either the 2' or 3'-hydroxyl group as the attachment site. The molecular basis for this flexibility was investigated. Introduction of the nucleotide U73 of tRNA(Cys) into tRNA(Val) was found to confer the flexibility. While valylation of the wild-type tRNA(Val) by the class I ValRS was strictly dependent on the terminal 2'-hydroxyl group, that of the U73 mutant of tRNA(Val) occurred at either the 2' or 3'-hydroxyl group. Thus, the single nucleotide U73 of tRNA has the ability to break the stereo barrier of amino acid attachment to tRNA, by mobilizing the 2' and 3'-hydroxyl groups of A76 in flexible geometry with respect to the tRNA acceptor stem.     

Impaired affinity for phenylalanine in Escherichia coli phenylalanyl-tRNA synthetase mutant caused by Gly-to-Asp exchange in motif 2 of class II tRNA synthetases

Phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 subunit structure) is a member of class II of tRNA synthetases. We report here the genetic analysis of an Escherichia coli mutant strain which is auxotrophic for phenylalanine because it has a PheRS with a decreased affinity for phenylalanine. The mutant pheS gene encoding the PheRS alpha subunit was cloned and sequenced, and the deviation from the wild-type gene was found to result in a Gly191-to-Asp191 exchange. This alteration is located within motif 2, one of 3 conserved sequence motifs characteristic for class II aminoacyl-tRNA synthetases. Motif 2 may thus participate in the formation of the phenylalanine binding site in PheRS.     

Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps

Comprehensive steady-state and transient kinetic studies of the synthetic and editing activities of Escherichia coli leucyl-tRNA synthetase (LeuRS) demonstrate that the enzyme depends almost entirely on post-transfer editing to endow the cell with specificity against incorporation of norvaline into protein. Among the three class I tRNA synthetases possessing a dedicated post-transfer editing domain (connective peptide 1; CP1 domain), LeuRS resembles valyl-tRNA synthetase in its reliance on post-transfer editing, whereas isoleucyl-tRNA synthetase differs in retaining a distinct tRNA-dependent synthetic site pre-transfer editing activity to clear noncognate amino acids before misacylation. Further characterization of the post-transfer editing activity in LeuRS by single-turnover kinetics demonstrates that the rate-limiting step is dissociation of deacylated tRNA and/or amino acid product and highlights the critical role of a conserved aspartate residue in mediating the first-order hydrolytic steps on the enzyme. Parallel analyses of adenylate and aminoacyl-tRNA formation reactions by wild-type and mutant LeuRS demonstrate that the efficiency of post-transfer editing is controlled by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA and shows that trans editing after rebinding is a competent kinetic pathway. Together with prior analyses of isoleucyl-tRNA synthetase and valyl-tRNA synthetase, these experiments provide the basis for a comprehensive model of editing by class I tRNA synthetases, in which kinetic partitioning plays an essential role at both pre-transfer and post-transfer steps.     

Crystal structures of Entamoeba histolytica lysyl-tRNA synthetase reveal conformational changes upon lysine binding and a specific helix bundle domain

The class II lysyl-tRNA synthetases (KRS) are conserved aminoacyl-tRNA synthetases that attach lysine to the cognate tRNA in a two-step mechanism. The enzyme from the parasitic protozoan Entamoeba histolytica was crystallized in the presence of small ligands to generate snapshots of the lysine-adenylate formation. The residues involved in lysine activation are highly conserved and the active site closes around the lysyl-adenylate, as observed in bacterial KRS. The Entamoeba EMAPII-like polypeptide is not resolved in the crystals, but another Entamoeba-specific insertion could be modeled as a small helix bundle that may contribute to tRNA binding through interaction with the tRNA hinge.     

Recent results on how aminoacyl transfer RNA synthetases recognize specific transfer RNAs

Summary Aminoacyl tRNA synthetases discriminate between tRNA species by a highly specific mechanism. Physical and chemical studies indicate that the synthetases bind along and around the inside of the three-dimensional L-shaped tRNA structure. Studies of mutant tRNAs that affect synthetase interaction tend to confirm this conclusion. However, in contrast to proteins that recognize a specific block of contiguous nucleotide units (e.g., repressors, restriction enzymes, etc.), synthetases appear to interact with spatially disperse elements of the structure. Available evidence suggests that tRNA binding clefts on various synthetases may be roughly similar, with specificity being achieved by the choice of amino acid residues in a few critical positions in the tRNA binding clefts. With this idea in mind, it should be possible to introduce amino acid substitutions into the binding clefts and thereby change tRNA recognition specificity. This has been attempted (by genetic manipulations) and a mutant alanine tRNA synthetase with altered tRNA recognition has been isolated. This enzyme can attach alanine to isoleucine specific tRNA. When presented with valine specific tRNA, a tRNA similar in some structural features to the isoleucine specific tRNA, or with the structurally quite different tyrosine specific tRNA, no significant aminoacylation occurs. Thus, a precise specificity alteration can occur through mutation; this result supports the idea of similarities in synthetase binding clefts, with specificity being achieved by the positioning of amino acids at critical positions in these clefts. Finally, further data have been obtained on the issue of possible transient covalent bond formation between synthetases and tRNAs, as a critical part of the interaction.Abbreviations tRNA^x a tRNA specific for the amino acid - x where x is given the standard 3 letter abbreviation     

Anticodon recognition in evolution: switching tRNA specificity of an aminoacyl-tRNA synthetase by site-directed peptide transplantation

The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of aminoacyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five beta-strands of the OB-fold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L45). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNALys (anticodon UUU) toward tRNAAsp (GUC) could be attempted by transplanting the small loop L45 of aspartyl-tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNALys. In exchange, the chimeric enzyme acquires the capacity to charge tRNAAsp with lysine. Upon giving the tRNAAsp substrate the discriminator base of tRNALys, the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys --> Asp mutation inserted into the beta-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases.