A succession of substrate induced conformational changes ensures the amino acid specificity of Thermus thermophilus prolyl-tRNA synthetase: comparison with histidyl-tRNA synthetase

We describe the recognition by Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) of proline, ATP and prolyl-adenylate and the sequential conformational changes occurring when the substrates bind and the activated intermediate is formed. Proline and ATP binding cause respectively conformational changes in the proline binding loop and motif 2 loop. However formation of the activated intermediate is necessary for the final conformational ordering of a ten residue peptide ("ordering loop") close to the active site which would appear to be essential for functional tRNA 3' end binding. These induced fit conformational changes ensure that the enzyme is highly specific for proline activation and aminoacylation. We also present new structures of apo and AMP bound histidyl-tRNA synthetase (HisRS) from T. thermophilus which we compare to our previous structures of the histidine and histidyl-adenylate bound enzyme. Qualitatively, similar results to those observed with T. thermophilus prolyl-tRNA synthetase are found. However histidine binding is sufficient to induce the co-operative ordering of the topologically equivalent histidine binding loop and ordering loop. These two examples contrast with most other class II aminoacyl-tRNA synthetases whose pocket for the cognate amino acid side-chain is largely preformed. T. thermophilus prolyl-tRNA synthetase appears to be the second class II aminoacyl-tRNA synthetase, after HisRS, to use a positively charged amino acid instead of a divalent cation to catalyse the amino acid activation reaction.     

tRNA discrimination by T. thermophilus phenylalanyl-tRNA synthetase at the binding step

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.     

The 2 A crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue

Leucyl-, isoleucyl- and valyl-tRNA synthetases are closely related large monomeric class I synthetases. Each contains a homologous insertion domain of approximately 200 residues, which is thought to permit them to hydrolyse ('edit') cognate tRNA that has been mischarged with a chemically similar but non-cognate amino acid. We describe the first crystal structure of a leucyl-tRNA synthetase, from the hyperthermophile Thermus thermophilus, at 2.0 A resolution. The overall architecture is similar to that of isoleucyl-tRNA synthetase, except that the putative editing domain is inserted at a different position in the primary structure. This feature is unique to prokaryote-like leucyl-tRNA synthetases, as is the presence of a novel additional flexibly inserted domain. Comparison of native enzyme and complexes with leucine and a leucyl- adenylate analogue shows that binding of the adenosine moiety of leucyl-adenylate causes significant conformational changes in the active site required for amino acid activation and tight binding of the adenylate. These changes are propagated to more distant regions of the enzyme, leading to a significantly more ordered structure ready for the subsequent aminoacylation and/or editing steps.     

Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus

The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.     

Two types of tRNA(Gm)methylase found in extreme thermophile, Thermus thermophilus strains HB 8 and HB 27

There are two distinct strains HB 8 and HB 27 in an extreme thermophile, Thermus thermophilus, and both strains have their own tRNA(Gm)methylases, which specifically methylates the 2'-OH of the ribose ring in the D loop of tRNA. The Gm-methylases are very similar with respect to the recognition mechanism of substrate tRNA and the molecular weight, but differ in the temperature dependency of the enzyme activity. Gm-methylase from strain HB 8 possesses its activity even at low temperature (40 degrees C), whereas that of strain HB 27 shows very low activity at the temperature and increases the activity as the incubation temperature is raised. Amino acid compositions of both the enzymes are very similar except for Glx and Asx, but the content of secondary structure is very different as judged by circular dichroism.     

Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition

Bacterial tyrosyl-tRNA synthetases (TyrRS) possess a flexibly linked C-terminal domain of approximately 80 residues, which has hitherto been disordered in crystal structures of the enzyme. We have determined the structure of Thermus thermophilus TyrRS at 2.0 A resolution in a crystal form in which the C-terminal domain is ordered, and confirm that the fold is similar to part of the C-terminal domain of ribosomal protein S4. We have also determined the structure at 2.9 A resolution of the complex of T.thermophilus TyrRS with cognate tRNA(tyr)(G Psi A). In this structure, the C-terminal domain binds between the characteristic long variable arm of the tRNA and the anti-codon stem, thus recognizing the unique shape of the tRNA. The anticodon bases have a novel conformation with A-36 stacked on G-34, and both G-34 and Psi-35 are base-specifically recognized. The tRNA binds across the two subunits of the dimeric enzyme and, remarkably, the mode of recognition of the class I TyrRS for its cognate tRNA resembles that of a class II synthetase in being from the major groove side of the acceptor stem.     

Antideterminants present in minihelix(Sec) hinder its recognition by prokaryotic elongation factor Tu.

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor. tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.     

The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing conformation

Leucyl-tRNA synthetase (LeuRS) has a specific post-transfer editing activity directed against mischarged isoleucine and similar noncognate amino acids. We describe the post-transfer-editing and product complexes of Thermus thermophilus LeuRS (LeuRSTT) with tRNA(Leu) at 2.9- to 3.3-A resolution. In the post-transfer-editing configuration, A76 binds in the editing active site exactly as previously found for the adenosine moiety of a small-molecule editing-substrate analog. The 60 C-terminal residues of LeuRSTT, unseen in previous structures, fold into a compact domain flexibly linked to the rest of the molecule and interacting with the G19-C56 tertiary base pair of tRNA(Leu). LeuRS recognition of tRNA(Leu) depends essentially on tRNA shape rather than base-specific interactions. The structures show that considerable domain rotations, notably of the editing domain, accompany the tRNA-3' end dynamics associated successively with aminoacylation, post-transfer editing and product release.     

A better enzyme to cope with cold. Comparative flexibility studies on psychrotrophic, mesophilic, and thermophilic IPMDHs

3-isopropylmalate dehydrogenase (IPMDH) from the psychrotrophic bacterium Vibrio sp. I5 has been expressed in Escherichia coli and purified. This cold-adapted enzyme is highly homologous with IPMDHs from other organisms, including mesophilic E. coli and thermophilic Thermus thermophilus bacteria. Its molecular properties are similar to these counterparts. Whereas the E. coli and T. thermophilus enzymes are hardly active at room temperature, the Vibrio IPMDH has reasonable activity below room temperature. The thermal stabilities, conformational flexibilities (hydrogen-deuterium exchange), and kinetic parameters of these enzymes were compared. The temperature dependence of the catalytic parameters of the three enzymes show similar but shifted profiles. The Vibrio IPMDH is a much better enzyme at 25 degrees C than its counterparts. With decreasing temperature i.e. with decreasing conformational flexibility, the specific activity reduces, as well; however, in the case of the Vibrio enzyme, the residual activity is still high enough for normal physiological operation of the organism. The cold-adaptation strategy in this case is achieved by creation of an extremely efficient enzyme, which has reduced but still sufficient activity at low temperature.     

Structures of argininosuccinate synthetase in enzyme-ATP substrates and enzyme-AMP product forms: stereochemistry of the catalytic reaction

Argininosuccinate synthetase reversibly catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate. The structures of the enzyme from Thermus thermophilus HB8 complexed with intact ATP and substrates (citrulline and aspartate) and with AMP and product (argininosuccinate) have been determined at 2.1- and 2.0-A resolution, respectively. The enzyme does not show the ATP-induced domain rotation observed in the enzyme from Escherichia coli. In the enzyme-substrate complex, the reaction sites of ATP and the bound substrates are adjacent and are sufficiently close for the reaction to proceed without the large conformational change at the domain level. The mobility of the triphosphate group in ATP and the side chain of citrulline play an important role in the catalytic action. The protonated amino group of the bound aspartate interacts with the alpha-phosphate of ATP and the ureido group of citrulline, thus stimulating the adenylation of citrulline. The enzyme-product complex explains how the citrullyl-AMP intermediate is bound to the active site. The stereochemistry of the catalysis of the enzyme is clarified on the basis of the structures of tAsS (argininosuccinate synthetase from T. thermophilus HB8) complexes.     

Fluorescence-monitored conformational change on the 3'-end of tRNA upon aminoacylation.

Fluorescent tRNAs species with formycine in the 3'-terminal position (tRNA-CCF) were derived from Escherichia coli tRNA(Val). Thermus thermophilus tRNA(Aap) and Thermus thermophilus tRNA(Phe). The fluorescence of formycine was used to monitor the conformational changes at the 3'-terminus of tRNA caused by aminoacylation and hydrolysis of aminoacyl residue from aminoacyl-tRNAs. An increase of about 15% in the fluorescence intensity was observed after aminoacylation of the three tRNA-CCF. This change in fluorescence amplitude that is reversed by hydrolysis of the aminoacyl residue, does not depend on the structure of the amino acid or tRNA sequence. A local conformational change at the 3'-terminal formycine probably involving a partial destacking of the base moiety in the ACCF end takes place as a consequence of aminoacylation. A structural change at the 3'-terminus of tRNA induced by attachment and detachment of the acyl residue may be important in controlling the substrate/product relationship in reactions in which tRNA participates during protein biosynthesis.     

Crystal structure of a eukaryote/archaeon-like protyl-tRNA synthetase and its complex with tRNAPro(CGG)

Prolyl-tRNA synthetase (ProRS) is a class IIa synthetase that, according to sequence analysis, occurs in different organisms with one of two quite distinct structural architectures: prokaryote-like and eukaryote/archaeon-like. The primary sequence of ProRS from the hypothermophilic eubacterium Thermus thermophilus (ProRSTT) shows that this enzyme is surprisingly eukaryote/archaeon-like. We describe its crystal structure at 2.43 angstom resolution, which reveals a feature that is unique among class II synthetases. This is an additional zinc-containing domain after the expected class IIa anticodon-binding domain and whose C-terminal extremity, which ends in an absolutely conserved tyrosine, folds back into the active site. We also present an improved structure of ProRSTT complexed with tRNAPro(CGG) at 2.85 angstom resolution. This structure represents an initial docking state of the tRNA in which the anticodon stem-loop is engaged, particularly via the tRNAPro-specific bases G35 and G36, but the 3' end does not enter the active site. Considerable structural changes in tRNA and/or synthetase, which are probably induced by small substrates, are required to achieve the conformation active for aminoacylation.     

Purification and characterization of tRNA(adenosine-1-)-methyltransferase from Thermus thermophilus HB27.

A58, the conserved adenosine residue in the T psi C loop of tRNAs, is methylated to m1A 58 in an extreme thermophile, Thermus thermophilus HB27. The enzyme catalyzing this methyltransfer reaction was purified from the thermophle. The substrate specificity of the enzyme was investigated by using tRNA fragments. The enzyme can transfer the methyl group to the 3'-half fragment of E. coli initiator tRNA, indicating that the main recognition site of the enzyme exists in the 3' half of tRNA including the T-loop and the T-stem.     

Aspartyl tRNA-synthetase from Escherichia coli: flexibility and adaptability to the substrates

The crystal structure of aspartyl-tRNA synthetase from Escherichia coli has been determined to a resolution of 2.7 A. The structure is compared to the same enzyme co-crystallized with tRNA(Asp) and containing aspartyl adenylate or ATP. The asymmetric unit contains three monomers of the enzyme. While most parts of the protein show no significant differences in the three monomers, a few regions cannot be superimposed. Those regions are characterized by a high B-factor, and consist mostly of loops that make contacts with the tRNA in the complexes. The flexibility of the protein is seen at a global level, by the observation of a 10 to 15 degrees rotation of the N-terminal and insertion domains upon tRNA binding, and at the level of the individual amino acid residues, by main-chain and side-chain rearrangements. In contrast to these induced-fit conformational changes, a few residues essential for the tRNA anticodon or aspartyl-adenylate recognition exist in a predefined conformation, ensured by specific interactions within the protein.     

tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase.

The ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., S?ll, D. & Weygand-Durasevic, I. (1997) J. Biol. Chem.272, 1136-1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl-adenylate formation than the apoenzyme alone. Sequence-specific tRNA-protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA-induced conformational change in the enzyme's active site. The 3'-terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA-assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.     

The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site.

The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl-tRNA synthetase with tRNA(Ser) (GGA) and a non-hydrolysable seryl-adenylate analogue has been refined at 2.7 angstrom resolution. The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits. The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem. In particular, a novel ring-ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs. This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA. Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition. These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps.     

Identification of two distinct hybrid state intermediates on the ribosome

High spatial and time resolution single-molecule fluorescence resonance energy transfer measurements have been used to probe the structural and kinetic parameters of transfer RNA (tRNA) movements within the aminoacyl (A) and peptidyl (P) sites of the ribosome. Our investigation of tRNA motions, quantified on wild-type, mutant, and L1-depleted ribosome complexes, reveals a dynamic exchange between three metastable tRNA configurations, one of which is a previously unidentified hybrid state in which only deacylated-tRNA adopts its hybrid (P/E) configuration. This new dynamic information suggests a framework in which the formation of intermediate states in the translocation process is achieved through global conformational rearrangements of the ribosome particle.     

Theoretical study of the structure of adenosine deaminase complexes with adenosine analogues: I. Aza-, deaza- and isomeric azadeazaanalogues of adenosine

The conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13 isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme (the 1ADD structure was used) within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of the ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1-deazaadenosine (1ADD) known from the X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of the bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.