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Non-discriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain

2003; Springer Nature; Volume: 22; Issue: 7 Linguagem: Inglês

10.1093/emboj/cdg148

1460-2075

Christophe Charron, Hervé Roy, Mickaël Blaise, Richard Giegé, Daniel Kern,

RNA modifications and cancer

Article1 April 2003free access Non-discriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain Christophe Charron Christophe Charron Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Hervé Roy Hervé Roy Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Mickael Blaise Mickael Blaise Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Richard Giegé Corresponding Author Richard Giegé Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Daniel Kern Daniel Kern Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Christophe Charron Christophe Charron Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Hervé Roy Hervé Roy Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Mickael Blaise Mickael Blaise Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Richard Giegé Corresponding Author Richard Giegé Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Daniel Kern Daniel Kern Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Author Information Christophe Charron1, Hervé Roy1, Mickael Blaise1, Richard Giegé 1 and Daniel Kern1 1Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, cedex, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1632-1643https://doi.org/10.1093/emboj/cdg148 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In most organisms, tRNA aminoacylation is ensured by 20 aminoacyl-tRNA synthetases (aaRSs). In eubacteria, however, synthetases can be duplicated as in Thermus thermophilus, which contains two distinct AspRSs. While AspRS-1 is specific, AspRS-2 is non-discriminating and aspartylates tRNAAsp and tRNAAsn. The structure at 2.3 Å resolution of AspRS-2, the first of a non-discriminating synthetase, was solved. It differs from that of AspRS-1 but has resemblance to that of discriminating and archaeal AspRS from Pyrococcus kodakaraensis. The protein presents non-conventional features in its OB-fold anticodon-binding domain, namely the absence of a helix inserted between two β-strands of this fold and a peculiar L1 loop differing from the large loops known to interact with tRNAAsp identity determinant C36 in conventional AspRSs. In AspRS-2, this loop is small and structurally homologous to that in AsnRSs, including conservation of a proline. In discriminating Pyrococcus AspRS, the L1 loop, although small, lacks this proline and is not superimposable with that of AspRS-2 or AsnRS. Its particular status is demonstrated by a loop-exchange experiment that renders the Pyrococcus AspRS non-discriminating. Introduction Aminoacyl-tRNA synthetases (aaRSs) constitute a family of enzymes of structural and functional diversity (Martinis et al., 1999; Ibba et al., 2003). Their pivotal role is in protein synthesis where they ensure translation of the genetic code into proteins. Correct translation relies on specific charging by each of the 20 synthetases of the cognate isoaccepting tRNAs with the homologous amino acid (First, 1998; Ibba and Söll, 2000). However, exceptions to the rule of unity of tRNA aminoacylation systems, namely one synthetase for each of the 20 amino acids, appeared with the discovery in some organisms of duplicated or missing synthetases (Becker and Kern, 1998; Ibba and Söll, 2001). This is the case in the thermophilic eubacterium Thermus thermophilus, which contains two genetically distinct aspartyl-tRNA synthetases (AspRSs) (Becker et al., 1997). The distinction is also functional, since AspRS-1 charges solely tRNAAsp in strong contrast to AspRS-2, which aspartylates both tRNAAsp and tRNAAsn with similar catalytic efficiencies, despite the presence in T.thermophilus of a fully functional asparaginyl-tRNA synthetase (AsnRS). Aspartate mischarged on tRNAAsn is then converted into asparagine by a tRNA-dependent amidotransferase. Altogether, this establishes coexistence in T.thermophilus of two distinct pathways of both tRNA aspartylation and tRNA asparaginylation. While AspRS-1 shares functional and structural features with other eubacterial AspRSs, the sequence of AspRS-2 strikingly resembles those of the non-discriminating AspRSs present in archaea lacking AsnRS (Curnow et al., 1996; Becker et al., 2000; Tumbula et al., 2000; Tumbula-Hansen et al., 2002). Thus, the two distinct systems, one archaeal-like and the other one eubacterial, are probably related to ancestral and modern aminoacylation pathways. This study addresses the question of the structural basis accounting for the relaxed specificity of the archaeal-type AspRS-2 from T.thermophilus. To this aim, the crystal structure of this protein belonging to class IIb synthetases was solved. It is the first one of a non-discriminating synthetase. Structural analysis reveals features that distinguish AspRS-2 from the crystal structure of AspRS-1 (Delarue et al., 1994; Ng et al., 2002) and from that of other AspRSs (Giegé and Rees, 2003). Despite its eubacterial origin, non-discriminating AspRS-2 presents architectural features found in eukaryotic (Ruff et al., 1991; Sauter et al., 2000) and archaeal (Schmitt et al., 1998) AspRSs. The most prominent difference from conventional AspRSs lies in the conformation of the anticodon-recognizing domain, and more precisely in that of two loops joining β-strands within its OB-fold, characteristic of class IIb synthetases (see Figure 6B). One shares strong conformational resemblance with a homologous loop present in AsnRSs. Altogether, the new structural data combined with functional experiments account for the relaxed tRNA recognition of AspRS-2 and shed new light on the structure–function relationship of archaeal-type and archaeal AspRSs. Results and discussion Structure determination Because of significant sequence similarities, the structure of native AspRS-2 was solved initially by molecular replacement using AspRS from the archeon Pyrococcus kodakaraensis (Schmitt et al., 1998) as the starting model. Since the quality of the electron density did not allow an easy trace of the protein fold in a few regions of the electron density map, the multiwavelength anomalous dispersion (MAD) method was used on a selenomethionine-substituted protein to ensure an unbiased structure. The final model of AspRS-2 was refined to an Rfactor of 22.7% and an Rfree of 26.2% between 30 and 2.3 Å resolution and has tightly restrained geometry (r.m.s. bond and angle deviations of 0.007 Å and 1.227°, respectively; Table I). As examples, Figure 1 shows the quality of the electron density map in the active site domain and anticodon-binding region of the enzyme. Over 91% of the residues are within the most favoured regions in a Ramachandran plot, as defined by PROCHECK (Laskowski et al., 1993). Only well-ordered Glu191 of each subunit has unfavourable main-chain torsion angles, as found in P.kodakaraensis AspRS for the equivalent acidic residue Asp204. Figure 1.Stereoviews of the MAD electron density map (contoured at 1.0 σ) of part of the catalytic site (A) and anticodon-binding (B) domains of T.thermophilus AspRS-2. In (A), the β-strands A2, A3, A4, A5, and A6 are labelled as in the P.kodakaraensis AspRS structure (Schmitt et al., 1998). In (B), displaying an amino acid stretch (residues 65–75) comprising loop L1, notice the functionally important Pro72 and the lack of density for the side chain of Lys70 (see text). Download figure Download PowerPoint Table 1. Crystallographic statistics Data collection and MAD phasing Native Se edge Se peak Se remote Space group P212121 Unit cell constants (Å) a = 57.4, b = 122.6, c = 167.1 Wavelength (Å) 0.9330 0.9798 0.9796 0.9150 Resolution (Å) 30–2.3 30–2.3 30–2.3 30–2.3 Completeness (%)a 99.8 (99.9) 98.5 (99.5) 99.8 (99.9) 96.5 (97.2) Rsym (%)a,b 4.5 (22.1) 5.3 (28.1) 6.1 (28.2) 5.3 (24.1) Multiplicitya 4.2 (4.4) 3.4 (3.4) 4.8 (4.8) 3.1 (3.1) I/σ(I)a 11.5 (3.1) 9.9 (2.2) 8.8 (2.5) 10.1 (2.9) Resolution (Å) 30–2.3 Reflections work set 45 048 Reflections test set 3443 Rfactor (%)c 22.7 Rfree (%)d 26.2 R.m.s.d. bonds (Å) 0.007 R.m.s.d. angles (°) 1.2 Mean B values (Å2) 38.1 No. of protein atoms 5769 No. of solvent molecules 160 a Number in parentheses corresponds to the last resolution shell 2.36–2.30 Å. b Rsym = Σ|I − |ΣI c Rfactor = Σ‖Fobs| − |Fcalc‖/Σ|Fobs| d For Rfree calculation, 7% of data were selected. The T.thermophilus AspRS structure visualizes residues 1–93, 111–150, 179–199 and 213–414 of subunit A, residues 1–97, 111–150, 180–199 and 213–414 of subunit B and 160 water molecules solvating the dimer. As already suspected in the model obtained by molecular replacement, a few amino acid stretches covering 14% of the AspRS-2 sequence are not seen in the final MAD model because of non-existent density. They correspond to the last nine C-terminal amino acids of the synthetase (residues 415–422) and to three loops. One non-defined loop is located in the hinge region connecting the anticodon-binding and active site domains (residues 98–110) and the two others are in the active site domain. They are a region comprising the so-called mobile flipping loop (as defined in Schmitt et al., 1998) (residues 151–178) and the loop of class II consensus motif 2 (residues 200–212). It is likely that these mobile loops will acquire fixed conformations when liganded with substrates. Further, some side chains of surface residues are not well defined (Lys9, Lys51, Lys70, Tyr111, Lys280, Glu305, Glu330, Glu361, Glu371); Figure 1B shows the example of Lys70 in the anticodon-binding domain of the synthetase. Mobile regions were observed in other class IIb synthetases. In the apo form of T.thermophilus AsnRS, the homologues of these loops only became defined when AsnRS was complexed with a non-hydrolysable analogue of asparaginyl-adenylate (Berthet-Colominas et al., 1998). In Escherichia coli LysRS (LysU) complexed with lysine, seven residues in the hinge region could not be built because of lack of density (Onesti et al., 1995; Desogus et al., 2000). Since this region is in contact with tRNAAsp in the yeast (Ruff et al., 1991; Cavarelli et al., 1993) and T.thermophilus (Briand et al., 2000) complexes, it is suggested that stabilization of its non-built homologue in AspRS-2 will be brought about by tRNA binding. The movement of the hinge domain in yeast AspRS, which allows binding of the tRNA D-stem, supports this view (Sauter et al., 2000). Overall description of the structure of non-discriminating AspRS-2 AspRS-2 from T.thermophilus is a homodimer with each subunit comprising 422 amino acids (Becker et al., 2000). The protein has a modular architecture. Its N-terminal β-sheet-rich anticodon-binding domain resembles an OB-fold as defined by Murzin (1993) and is linked by a short interconnection to a C-terminal active-site domain, comprising the three class II consensus motives, which is built around a six-stranded antiparallel β-sheet surrounded by α-helices (Figure 2A). This architecture is characteristic of class IIb synthetases. Figure 2.Structure of T.thermophilus AspRS-2. (A) Ribbon representation of the dimeric synthetase. Subunit A is drawn in yellow (N-terminal domain) and in orange (catalytic domain), and subunit B is in blue (N-terminal domain) and purple (catalytic domain). The N- and C-terminal ends of each subunit are labelled. (B) Electrostatic potential mapped on the molecular surface of dimeric AspRS-2, as computed with Swiss-Pdb Viewer (Guex and Peitsch, 1997). Blue, white and red regions correspond to positive, neutral and negative electrostatic potentials, respectively. The putative location of a backbone model of tRNAAsp, as in the complex with T.thermophilus AspRS-1 (Briand et al., 2000), covering 'blue' regions of positive potential, is indicated. The orientation of the synthetase is as in (A). Download figure Download PowerPoint AspRS-2 has a dimeric interface surface of 5829 Å2, the largest interface known so far in an AspRS (to be compared with 4600 Å2, the smallest interface as found in yeast AspRS; see Sauter et al., 2001). The structure is well defined in the anticodon-binding domain but contains a few disordered regions in the rest of the molecule. When analysing these regions, faint differences appear between the two subunits. The most prominent one concerns residues 94–97 in the hinge domain, which could be traced in one subunit and are not seen in the other one. Indeed, the connection between active site and anticodon-binding domains is only partly seen in the electron density map of AspRS-2. Of the 24 amino acids making the connection, only seven upstream of the active-site domain of both subunits and four downstream of the anticodon-binding domain in subunit B are seen. Altogether, this is indicative of the pseudo-homodimeric nature of AspRS-2, reflected by the conformational heterogeneity of the monomers. The differences can be quantitated by superimposition of the main-chain atoms of subunits A and B of the dimer and correspond to an r.m.s. deviation of 0.424 Å. Calculation of the electrostatic potential reveals two symmetrical positive zones at the molecular surface of the AspRS-2 dimer, which span from the anticodon-binding domain to the catalytic centre of the enzyme. They are similar to the footprints of tRNAAsp on T.thermophilus AspRS-1, as deduced from the crystal structure of the complex (Briand et al., 2000), and thus most likely correspond to the binding areas of tRNA (Figure 2B). Structural and functional comparison of AspRS-2 with other AspRSs of known crystal structure The structural view. A sequence alignment based on the superimposition of the Cα traces in AspRS-2 and in the four other known crystallographic structures of AspRSs shows 49 strictly conserved residues (Figure 3A). Overall, the comparison reveals a greater relatedness of AspRS-2 with AspRS of P.kodakaraensis than with other AspRSs (e.g. in the anticodon-binding and hinge domains). This relatedness is clearly seen in the phylogenetic tree of AspRSs (Figure 3B). Figure 3.Structure-based sequence alignment of AspRSs of known crystal structure and simplified phylogenetic tree of AspRSs. (A) Subdomains in AspRS-2 showing structural deviations with P.kodakaraensis AspRS are emphasized on a green background (Figure 5B). Strictly conserved residues in the five crystal structures are on a red background (only Leu237 is not highly conserved); semi-conserved residues are in red; (−) missing residues. Residues in AspRS-2 not built in the crystal structure because of lack of density are in light grey. Strategic regions (see text) are boxed. Numbering corresponds to the AspRS-2 sequence (Becker et al., 2000). (B) The phylogenetic tree is adapted from Becker et al. (2000) and Woese et al. (2000). Notice that AsnRSs arise from the archaeal genre of AspRSs. Organisms are abbreviated as follows: D.r. (D.radiodurans), E.c. (E.coli), P.k. (P.kodakaraensis), S.c. (Saccharomyces cerevisiae) and T.t. (T.thermophilus); D.r.1 or 2 and T.t.1 or 2 refer to the eubacterial or archaeal forms of AspRS. Sequences are retrieved from DDBJ/EMBL/GenBank. Download figure Download PowerPoint Among the 36 conserved residues in the active site domain, eight were shown to ensure specific aspartate binding in the AspRSs from yeast (Cavarelli et al., 1994), E.coli (Eiler et al., 1999), T.thermophilus (Poterzman et al., 1994) and P.kodakaraensis (Schmitt et al., 1998). They are Glu158, Gln180, Lys183, Arg201, Asp218, Glu220, Glu345 and Arg352 (with the numbering of AspRS-2 sequence). For instance in P.kodakaraensis AspRS, Lys183 and Arg352 hydrogen bond with the aspartate carboxylate group, which is further stabilized by Arg201, Asp218 and Glu220. Given the conservation of these important functional amino acids, it is likely that recognition of aspartate by AspRS-2 occurs as in the four other AspRSs. Notice, however, in AspRS-2 the flexibility of Glu158 and of class IIb invariant Arg201, which are not seen, but are clearly visible in the apo form of other AspRSs (Eiler et al., 1999; Sauter et al., 2000; Ng et al., 2002). In the N-terminal domain, three conserved residues (Phe33, Gln44 and Glu76) participate in recognition of anticodon bases G34 and U35 by discriminating yeast (Cavarelli et al., 1993) and E.coli (Briand et al., 2000; Moulinier et al., 2001) AspRSs. Here also, it is likely that AspRS-2 recognizes these two bases, as do discriminating AspRSs. Likewise, the aromatic ring of Phe33 would stack the ring of U35 while side chains of Gln44 and Glu76 would interact with tRNAAsp via hydrogen bonds to U35 and G34, respectively. Several residues in AspRS-2 are only conserved in AspRSs originating from one or two of the three phylogenetic kingdoms of life. For instance, Ser348 is specific to archaeal and eukaryotic AspRSs and was shown to participate in stabilization of the transition state of the tRNA aspartylation reaction in the yeast and Pyrococcus enzymes (Cavarelli et al., 1994; Schmitt et al., 1998). This residue is absent in most eubacterial AspRSs. On the other hand, some conserved residues in most AspRSs are absent in AspRS-2. This is the case for Tyr214, which position in class II synthetases is generally occupied by a phenylalanine that stacks to the adenine ring of ATP (Cavarelli et al., 1994; Eiler et al., 1999). The absence of a phenylalanine at this position was also reported for hamster class IIa HisRS (Delarue and Moras, 1993) and P.kodakaraensis AspRS (Schmitt et al., 1998). Unlike in AspRS-2, this residue is almost always a hydrophobic amino acid (alanine, isoleucine or valine) in all known archaeal AspRS sequences (Becker et al., 2000). In contrast to other eubacterial AspRSs, AspRS-2 contains neither a C-terminal extension nor a large insertion domain between motifs 2 and 3 in its active site module (which would correspond to residues 296–391 in T.thermophilus AspRS-1). By these features it resembles archaeal and eukaryotic AspRSs. However, and in contrast to eukaryotic class IIb synthetases, AspRS-2 is missing the N-terminal extension upstream of the anticodon-binding domain. In yeast, this extension helps to bind the anticodon stem of tRNAAsp on the AspRS core (Frugier et al., 2000). In summary, non-discriminating eubacterial AspRS-2 reveals hybrid eukaryal and archaeal characteristics that are found in both its catalytic and its anticodon-binding domains. The functional view. Table II illustrates that AspRS-2 from T.thermophilus is non-discriminating and aspartylates tRNAAsp and tRNAAsn with comparable catalytic efficiency. This peculiar functionality is likely to rely on the relatedness of aspartate and asparagine identity sets that share the same discriminator base (G73) and similar GUC/U anticodons differing only by base 36 (C in tRNAAsp and U in tRNAAsn) (Giegé et al., 1998). As a likely consequence, the non-discriminating or discriminating nature of AspRSs should be linked to alternative recognition patterns of identity base 36. Table 2. Aspartylation of T.thermophilus tRNAAsp and tRNAAsn by native or engineered AspRSs AspRSs tRNAAsp charging tRNAAsn charging kcat s−1 (×10−2) Km μM kcat/Km (relative) kcat s−1 (×10−2) Km μM kcat/Km(relative)> T.t.2-nativea 4.2 0.14 1 0.56 0.20 0.09 P.k.-native 1.17 0.2 0.17 no detectable charging P.k.-mutantb 0.83 1.0 0.03 0.08 0.90 0.003 Aminoacylation conditions are as described in Materials and methods. Km and kcat values were determined at 37°C. a At 70°C, the kcat/Km for tRNA is only 2-fold higher than for tRNA (Becker and Kern, 1998). b The Pyrococcus mutant has the L1 loop exchanged with that from Thermus AspRS-2. As to the enzyme level, it is known that AspRSs recognize aspartate identity determinants and in particular anticodon determinant C36 (Giegé and Rees, 2003). In archaeal P.kodakaraensis AspRS, identification of a peculiar loop homologous to those recognizing the third anticodon base in other AspRSs led to the suggestion that this synthetase should be able to accommodate either a GUC or a GUU anticodon (Schmitt et al., 1998). Further, the conformational resemblance of non-discriminating AspRS-2 with archaeal AspRSs and the lack of AsnRS in some archaea, suggests that archaeal AspRSs, including AspRS from P.kodakaraensis, would be non-discriminating. Aminoacylation assays and biochemical characterization of the charged tRNAs, however, indicate that the Pyrococcus enzyme is discriminating and solely charges tRNAAsp (Figure 4A; Table II). This conclusion was also reached by others as the result of genome analysis and functional assays (Tumbula-Hansen et al., 2002). In addition, these authors showed the existence of both discriminating and non-discriminating forms of AspRS among the archaea, despite the high sequence identity of archaeal AspRSs. Following these considerations, the non-discriminating nature of AspRS-2 should be searched among the structural similarities and differences it has with Pyrococcus AspRS (and with other archaeal AspRSs), with the expectation that the features important for dual tRNA recognition are located in the anticodon-binding domain. Figure 4.Specificity of tRNA charging of T.thermophilus AspRSs and archaeal P.kodakaraensis AspRS and effect of the L1 loop on tRNA discrimination. (A) Aspartylation levels (plateau values) of unfractionated T.thermophilus tRNA with AspRS-1 or AspRS-2 and with native or mutated AspRS from Pyrococcus. (B) Identification by hybridization assays of tRNAAsp (a) and tRNAAsn (b) aspartylated by the various AspRSs. The AspRS mutant from P.kodakaraensis has the L1 loop exchanged by that from Thermus AspRS-2; C, control with uncharged tRNA. Download figure Download PowerPoint Similarities and differences between Thermus AspRS-2 and Pyrococcus AspRS Overall comparison. Comparison of AspRS-2 with AspRS from P.kodakaraensis reveals 41% sequence identity. This high level of identity suggests an overall conformational similarity of the two synthetases and has justified initial use of molecular replacement for solving the structure of AspRS-2. However, comparison of the two structures yields an r.m.s. deviation of superimposed Cα positions of 2.196 Å. This high value is indicative of conformational differences between the two proteins (Figure 5A), a conclusion in apparent contradiction with the high sequence identity. The contradiction is explained when comparing individual domains of the modular AspRS monomers. For the catalytic domain the r.m.s. deviation is reduced to 1.522 Å (Figure 5B) while for the anticodon-binding domains it becomes 1.260 Å (Figure 6A). The higher r.m.s. deviation for the entire monomer originates from a rigid-body movement of the anticodon-binding domain. Altogether, most residues in individual domains superimpose well, with deviations in the ∼1 Å range, but several local conformational changes, with structural deviations that can reach 6 Å, are identified at the protein surfaces (Figures 5 and 6A). Figure 5.Comparison of the 3D models of T.thermophilus AspRS-2 and of P.kodakaraensis AspRS (ATP form, Schmitt et al., 1998). (A) Superimposition of subunit A of AspRS-2 (green) and subunit A from P.kodakaraensis AspRS (red). Surface residues of the AspRS-2 active-site domain that show largest structural deviations with P.kodakaraensis AspRS are in italics. (B) Values of r.m.s. deviations calculated by least squares minimized superimposition of the active-site domains. Notice that the N-terminal domain of AspRS-2 deviates from that of P.kodakaraensis AspRS by a rigid body rotation of 4°. Download figure Download PowerPoint Figure 6.Comparison of anticodon-binding domains in T.thermophilus AspRS-2, P.kodakaraensis AspRS and T.thermophilus AsnRS. (A) Superimposition of the two domains in Thermus (green) and Pyrococcus (red) AspRSs (left) and r.m.s. deviations (right). (B) Ribbon representation of the anticodon-binding domain in AspRS-2 (left), Pyrococcus AspRS (middle) and Thermus AsnRS (right). Notice in Pyrococcus AspRS and in Thermus AsnRS a standard OB-fold formed by a five-stranded β-barrel with an α-helix (Hα displayed in red) between strands S3 and S4. In AspRS-2, α-helix Hα is replaced by the Lα loop. Loops and strands are coloured in yellow and green, respectively; Lα and L1 loops that are specific to AspRS-2 are emphasized by red labels (notice that L1 corresponds to L45 in the conventional OB-fold nomenclature; Murzin, 1993). Download figure Download PowerPoint Active-site domain. Structural differences between AspRS-2 and Pyrococcus AspRS are found in five regions centred on residues 188, 261, 288, 330 and 366 (Figure 5B). Four differences are due to insertions or deletions in sequence patches around residues 188, 261, 288 and 330 and lead to distortions of between 3 and 8 Å (Figures 3 and 5B). That occurring around Gly188 in AspRS-2 (Ala200 in Pyrococcus) near the N-terminus of β-sheet A2 of the catalytic domain, comprises deletion of the homologue to Ser201 in Pyrococcus AspRS. This deletion may affect the overall flexibility of the region comprising the flipping-loop. The three other differences concern regions at the AspRS surfaces that are not involved in catalysis, namely around Pro261 (Leu273 in Pyrococcus), Gly288 (Gly302 in Pyrococcus) and Glu330 (Lys346 in Pyrococcus). Sequence inspection shows that these insertions or deletions are characteristics of AspRS-2. Their putative role remains to be deciphered. Two other differences concern regions not seen in AspRS-2 because of high flexibility in the absence of bound ligands, but visible under different conformations in several crystal forms of Pyrococcus AspRS corresponding to specific functional states of the protein (Schmitt et al., 1998). One, with conserved Glu158, comprises the flipping-loop that anchors the aspartate substrate in the catalytic pocket. The other, centred on Glu203 and class II conserved Arg201, is the loop joining strands A2 and A3 in motif 2. It is involved in aminoacyl-adenylate formation and tRNA acceptor stem recognition. Interestingly, when comparing the structure of free or substrate-bound AspRSs from E.coli or yeast, the main conformational changes in the active-site domain concern precisely the flipping-loop and the loop of motif 2 (Rees et al., 2000; Sauter et al., 2000). A last conformational change corresponds to a global movement of a helix and part of a loop and occurs in AspRS-2 in the region comprising Lys366 (Lys382 in Pyrococcus). In subunit A of AspRS-2, this region is involved in a packing contact that does not occur in subunit B. Therefore, even if partly resulting from packing effects, it is likely that the conformation in the 360–370 region is an intrinsic characteristic of AspRS-2. Articulation between active site and anticodon-binding domains. Flexibility of the hinge domain, leading to different relative orientations of the two functional protein modules, is a characteristic of AspRSs and was shown to be associated with tRNA binding. In yeast AspRS the initial recognition of the tRNA anticodon loop necessitates a 6° rotation of the anticodon-binding domain relative to the active-site domain, so as to prevent a steric clash between the tRNA and the hinge domain of the synthetase (Sauter et al., 2000). Similar pathways were proposed for T.thermophilus AspRS-