M. A. Tukalo

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 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.

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.

Crystals of threonyl-tRNA synthetase from Thermus thermophilus. Preliminary crystallographic data.

Crystals have been obtained of threonyl-tRNA synthetase from the extreme thermophile Thermus thermophilus using sodium formate as a precipitant. The crystals are very stable and diffract to at least 2.4 A. The crystals belong to space group P2(1)2(1)2(1) with cell parameters a = 61.4 A, b = 156.1 A, c = 177.3 A.