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

Tukalo, M. A.; Yaremchuk, A.; Fukunaga, Ryuya; Yokoyama, Shigeyuki; Cusack, Stephen

doi:10.1038/nsmb986

Cited by 145 publications

(264 citation statements)

References 31 publications

Supporting

Mentioning

250

Contrasting

Unclassified

Order By: Relevance

“…In addition, a novel C-terminal domain has been proposed to be important for tRNA interactions (9)(10)(11)(12).…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Unique Insert of Leucyl-tRNA Synthetase Is Required for Aminoacylation and Not Amino Acid Editing

Martinis

2007

Biochemistry

View full text Add to dashboard Cite

Leucyl-tRNA synthetase (LeuRS) is a class I enzyme, which houses its aminoacylation active site in a canonical core that is defined by a Rossmann nucleotide binding fold. In addition, many LeuRSs bear a unique polypeptide insert comprised of about 50 amino acids located just upstream of the conserved KMSKS sequence. The role of this leucine-specific domain (LS-domain) remains undefined. We hypothesized that this domain may be important for substrate recognition in aminoacylation and/or amino acid editing. We carried out a series of deletion mutations and chimeric swaps within the leucine-specific domain of Escherichia coli. Our results support that the leucinespecific domain is critical for aminoacylation, but not required for editing activity. Kinetic analysis determined that deletion of the LS-domain primarily impacts k cat . Because of its proximity to the aminoacylation active site, we propose that this domain interacts with the tRNA during amino acid activation and/or tRNA aminoacylation. Although the leucine-specific domain does not appear to be important to the editing complex, it remains possible that it aids the dynamic translocation process that moves tRNA from the aminoacylation to the editing complex.Aminoacylation is catalyzed by an ancient family of enzymes called the aminoacyl-tRNA synthetases (aaRS) (1,2). It is important to translation that the aaRS correctly link amino acid to the corresponding tRNA acceptors. Each of the aaRS is responsible for covalently attaching one of twenty standard amino acids to a set of cognate tRNA isoacceptors. The esterification of amino acid to tRNA occurs via a two-step reaction:The first reversible step involves the activation of an amino acid concomitant with ATP hydrolysis to form an aminoacyl-adenylate intermediate. The amino acid is then transferred from the adenylate intermediate to the 3′ terminal adenosine moiety of the tRNA. The charged aminoacyl-tRNA binds to EF-Tu and is transported to the ribosome to extend the polypeptide chain.Leucyl-tRNA synthetase (LeuRS) is a class I aaRS, and is responsible for accurately aminoacylating leucine to its cognate tRNA Leu isoacceptors (3). As is characteristic of all class I synthetases, the catalytic core is defined by a Rossmann nucleotide binding fold (4) that comprises the main body of the enzyme (5). This canonical class I core of LeuRS contains inserts and appendages (5-7). For example, the enzyme has an amino acid editing function that † This work was supported by the National Institutes of Health (GM63789). resides within a large insert called the connective polypeptide 1 (CP1) (8). In addition, a novel C-terminal domain has been proposed to be important for tRNA interactions (9-12).One unique small insert called the leucine-specific domain (LS-domain) is found in many bacterial LeuRSs (5). Its function remains undefined. This compact domain that is comprised of five β-strands and two short α-helices flanks the entrance of the aminoacylation active site (5). The LS-domain is inserted after the last β-...

show abstract

“…In addition, a novel C-terminal domain has been proposed to be important for tRNA interactions (9)(10)(11)(12).…”

mentioning

confidence: 99%

“…A large ensemble of LeuRS co-crystal structures that include tRNA complexes in the aminoacylation and editing conformation have failed to suggest a specific role for the LSdomain (5,9,(13)(14)(15)(16). The co-crystal structure of P. horikoshii LeuRS with the tRNA in the aminoacylation conformation has been solved (13).…”

mentioning

confidence: 99%

A Unique Insert of Leucyl-tRNA Synthetase Is Required for Aminoacylation and Not Amino Acid Editing

Martinis

2007

Biochemistry

View full text Add to dashboard Cite

show abstract

“…This finding also weakens an alternative hypothesis that proposes that the unusual inhibitory properties of TM84 are caused by the inhibitor binding to an alternative site on the enzyme other than the synthetic active site 8,33 . The most striking feature of the TM84 ternary structure is the pronounced movement of the highly conserved KMSKS loop across the active site of the enzyme when compared with previous LeuRS Tt structures bound to adenylate analogues 5,8 or tRNA 6 (Fig. 4d).…”

Section: Tm84 Is a Tight-binding Inhibitor Of Trna Leu Aminoacylationmentioning

confidence: 83%

“…3). LeuRSs are also composed of a number of additional domains involved in binding tRNA Leu or in proofreading activities [4][5][6][7] . X-ray crystal structures have been obtained for LeuRSs from both archaeal and bacterial origins in complex with a number of substrates and substrate analogues, including Leu 5 and tRNA Leu (refs 6,7), a reaction-intermediate analogue of , and also substrate mimics of both postand pre-transfer editing reactions 8 .…”

mentioning

confidence: 99%

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

et al. 2013

Self Cite

View full text Add to dashboard Cite

Leucyl-tRNA synthetases (LeuRSs) have an essential role in translation and are promising targets for antibiotic development. Agrocin 84 is a LeuRS inhibitor produced by the biocontrol agent Agrobacterium radiobacter K84 that targets pathogenic strains of A. tumefaciens, the causative agent of plant tumours. Agrocin 84 acts as a molecular Trojan horse and is processed inside the pathogen into a toxic moiety (TM84). Here we show using crystal structure, thermodynamic and kinetic analyses, that this natural antibiotic employs a unique and previously undescribed mechanism to inhibit LeuRS. TM84 requires tRNA Leu for tight binding to the LeuRS synthetic active site, unlike any previously reported inhibitors. TM84 traps the enzyme-tRNA complex in a novel 'aminoacylation-like' conformation, forming novel interactions with the KMSKS loop and the tRNA 3 0 -end. Our findings reveal an intriguing tRNAdependent inhibition mechanism that may confer a distinct evolutionary advantage in vivo and inform future rational antibiotic design.

show abstract

“…Crystallographic structures of the T. thermophilus LeuRS complexed with a small molecule inhibitor bound to the synthetic active site (19) and tRNA Leu in the post-transfer editing configuration (39) reveal the existence of a potentially mobile flap that contains the crucial K600 and L570 residue in hs mt and E. coli LeuRS respectively. This flexible closing domain (residues 577-634 in T. thermophilus), also called the leucyl-specific domain, is located just before the catalytically important KMSKS motif (32) (Figure 1).…”

Section: Roles Of a Flexible Domain Containing A Critical Residue In mentioning

confidence: 99%

A Single Residue in Leucyl-tRNA Synthetase Affecting Amino Acid Specificity and tRNA Aminoacylation

Lue

2007

Biochemistry

View full text Add to dashboard Cite

Human mitochondrial leucyl-tRNA synthetase (hs mt LeuRS) achieves high aminoacylation fidelity without a functional editing active site, representing a rare example of a class I aminoacyl-tRNA synthetase (aaRS) that does not proofread its products. Previous studies demonstrated that the enzyme achieves high selectivity by using a more specific synthetic active site that is not prone to errors under physiological conditions. Interestingly, the synthetic active site of hs mt LeuRS displays a high degree of homology with prokaryotic, lower eukaryotic and other mitochondrial LeuRSs that are less specific. However, there is one residue that differs between hs mt and Escherichia coli (E. coli) LeuRSs located on a flexible closing loop near the signature KMSKS motif. Here we describe studies indicating that this particular residue (K600 in hs mt LeuRS and L570 in E. coli LeuRS) strongly impacts aminoacylation in two ways -it affects both amino acid discrimination and transfer RNA (tRNA) binding. While this residue may not be in direct contact with the amino acid or tRNA substrate, substitutions of this position in both enzymes leads to altered catalytic efficiency and perturbations to the discrimination of leucine and isoleucine. In addition, tRNA recognition and aminoacylation is affected. These findings indicate that the conformation of the synthetic active site -modulated by this residue -may be coupled to specificity and provide new insights into the origins of selectivity without editing.Aminoacyl-tRNA synthetases (aaRSs) 1 are important translational factors. They catalyze the covalent attachment of amino acids to their cognate tRNAs, an essential step in the translation of the genetic code (1-3). The fidelity of protein synthesis is dependent upon the accuracy with which aaRSs discriminate between cognate and noncognate amino acids and numerous cellular tRNAs.To explain the high fidelity for cognate amino acids exhibited by aaRSs, a "double sieve" model has been proposed. This model, relevant mainly to class I aaRSs, relies on the use of two functionally independent active sites to achieve amino acid selectivity (4,5). In the first synthetic active site, amino acids are recognized, activated with ATP, converted to aminoacyl adenylates, and then transferred to tRNA. Amino acids larger than the cognate substrate are excluded from this site by sterics; smaller amino acids present a more significant problem as they can be bound, misactivated, and used erroneously to acylate tRNA. To resolve these errors, some aaRSs utilize a second editing active site that proofreads the products made by the activation site and hydrolytically cleaves substrates containing non-cognate amino acids. Both pre-transfer editing of misactivated aminoacyl adenylates and post-transfer editing of misacylated tRNA can occur at this second active site. Many systems are severely affected by *To whom correspondence should be addressed: Phone: 617-552-3121, Fax: 617-552-2705, E-mail: shana.kelley@bc.edu. Many previous studies have focused ...

show abstract

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

Cited by 145 publications

References 31 publications

A Unique Insert of Leucyl-tRNA Synthetase Is Required for Aminoacylation and Not Amino Acid Editing

A Unique Insert of Leucyl-tRNA Synthetase Is Required for Aminoacylation and Not Amino Acid Editing

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

A Single Residue in Leucyl-tRNA Synthetase Affecting Amino Acid Specificity and tRNA Aminoacylation

Contact Info

Product

Resources

About