Anna Yaremchuk

The 2 Å 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 ∼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 Å 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.

Structural and Mechanistic Basis of Pre- and Posttransfer Editing by Leucyl-tRNA Synthetase

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

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 ∼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 Å reso lution 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 Å resolution of the complex of T.thermophilus TyrRS with cognate tRNAtyr(GΨA). 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 Ψ‐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.

The crystal structures of T. thermophilus lysyl-tRNA synthetase complexed with E. coli tRNA(Lys) and a T. thermophilus tRNA(Lys) transcript: anticodon recognition and conformational changes upon binding of a lysyl-adenylate analogue.

The crystal structures of Thermus thermophilus lysyl‐tRNA synthetase, a class IIb aminoacyl‐tRNA synthetase, complexed with Escherchia coli tRNA(Lys)(mnm5 s2UUU) at 2.75 A resolution and with a T. thermophilus tRNA(Lys)(CUU) transcript at 2.9 A resolution are described. In both complexes only the tRNA anticodon stem‐loop is well ordered. The mode of binding of the anticodon stem‐loop to the N‐terminal beta‐barrel domain is similar to that previously found for the homologous class IIb aspartyl‐tRNA synthetase‐tRNA(Asp) complex except in the region of the wobble base 34 where either mnm5 s2U or C can be accommodated. The specific recognition of the other anticodon bases, U‐35 and U‐36, which are both major identity elements in the lysine system, is also described. Additional crystallographic data on a ternary complex with a lysyl‐adenylate analogue show that binding of the intermediate induces significant conformational changes in the vicinity of the active site of the enzyme.

The crystal structure of leucyl-tRNA synthetase complexed with tRNA Leu 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 tRNALeu at 2.9- to 3.3-Å 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 tRNALeu. LeuRS recognition of tRNALeu 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.

tRNAPro anticodon recognition by Thermus thermophilus prolyl-tRNA synthetase

BACKGROUND Most aminoacyl-tRNA synthetases (aaRSs) specifically recognize all or part of the anticodon triplet of nucleotides of their cognate tRNAs. Class IIa and class IIb aaRSs possess structurally distinct tRNA anticodon-binding domains. The class IIb enzymes (LysRS, AspRS and AsnRS) have an N-terminal beta-barrel domain (OB-fold); the interactions of this domain with the anticodon stem-loop are structurally well characterised for AspRS and LysRS. Four out of five class IIa enzymes (ProRS, ThrRS, HisRS and GlyRS, but not SerRS) have a C-terminal anticodon-binding domain with an alpha/beta fold, not yet found in any other protein. The mode of RNA binding by this domain is hitherto unknown as is the rationale, if any, behind classification of anticodon-binding domains for different aaRSs. RESULTS The crystal structure of Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) in complex with tRNA(Pro) has been determined at 3.5 A resolution by molecular replacement using the native enzyme structure. One tRNA molecule, of which only the lower two-thirds is well ordered, is found bound to the synthetase dimer. The C-terminal anticodon-binding domain binds to the anticodon stem-loop from the major groove side. Binding to tRNA by ProRSTT is reminiscent of the interaction of class IIb enzymes with cognate tRNAs, but only three of the anticodon-loop bases become splayed out (bases 35-37) rather than five (bases 33-37) in the case of class IIb enzymes. The two anticodon bases conserved in all tRNA(Pro), G35 and G36, are specifically recognised by ProRSTT. CONCLUSIONS For the synthetases possessing the class IIa anticodon-binding domain (ProRS, ThrRS and GlyRS, with the exception of HisRS), the two anticodon bases 35 and 36 are sufficient to uniquely identify the cognate tRNA (GG for proline, GU for threonine, CC for glycine), because these amino acids occupy full codon groups. The structure of ProRSTT in complex with its cognate tRNA shows that these two bases specifically interact with the enzyme, whereas base 34, which can be any base, is stacked under base 33 and makes no interactions with the synthetase. This is in agreement with biochemical experiments which identify bases 35 and 36 as major tRNA identity elements. In contrast, class IIb synthetases (AspRS, AsnRS and LysRS) have a distinct anticodon-binding domain that specifically recognises all three anticodon bases. This again correlates with the requirements of the genetic code for cognate tRNA identification, as the class IIb amino acids occupy half codon groups.

Crystal structure of a eukaryote/archaeon-like prolyl-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 Å 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 Å 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.

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon–anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the ‘GTP’- and ‘GDP’-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

Crystallization and preliminary crystallographic analysis of Thermus thermophilus leucyl-tRNA synthetase and its complexes with leucine and a non-hydrolysable leucyl-adenylate analogue.

Leucyl-tRNA synthetase from Thermus thermophilus (LeuRSTT) is the first LeuRS to be crystallized. Two crystal forms of the native enzyme have been obtained using the hanging-drop vapour-diffusion method with ammonium sulfate as a precipitant. Crystals of the first form belong to space group I422 and have unit-cell parameters a = b = 312.4, c = 100.4 A. They diffract anisotropically to 3.5 A resolution in the c-axis direction and to only 6 A resolution in the perpendicular direction. Crystals of the second form, which can be obtained native or with leucine or a leucyl-adenylate analogue bound, belong to space group C222(1) and have unit-cell parameters a = 102. 4, b = 154.1, c = 174.3 A. They diffract to 1.9 A resolution and contain one monomer in the asymmetric unit. Selenomethionated LeuRSTT has been produced and crystals of the second form suitable for MAD analysis have been grown.

Crystallization of the seryl-tRNA synthetase-tRNASer complex from Thermus thermophilus

The complex between seryl-tRNA synthetase and its cognate tRNA from the extreme thermophile Thermus thermophilus has been crystallized from ammonium sulphate solutions. Two different tetragonal crystal forms have been characterized, both diffracting to about 6 A using synchrotron radiation. One form grows as large bipyramids and has cell dimensions a = b = 127 A, c = 467 A, and the second form occurs as long, thin square prisms with cell dimensions a = b = 101 A, c = 471 A. Analysis of washed and dissolved crystals demonstrates the presence of both protein and tRNA.