Structures of Tryptophanyl-tRNA Synthetase II from Deinococcus radiodurans Bound to ATP and Tryptophan

Abstract

An auxiliary tryptophanyl tRNA synthetase (drTrpRS II) that interacts with nitric-oxide synthase in the radiation-resistant bacterium Deinococcus radiodurans charges tRNA with tryptophan and 4-nitrotryptophan, a specific nitration product of nitric-oxide synthase. The most critical molecular recognition events in the translation of the genetic code involve the error-free attachment of 21 amino acids to tRNAs that bear the appropriate anticodon triplets (1-3). These reactions are catalyzed by aminoacyltRNA synthetases (AARSs), 1 which are divided into two classes (I and II) based on their structures and reactivities (4). Class I synthetases attach amino acids to the 2Ј-hydroxyl group of the 3Ј-terminal adenosine of tRNA and comprise a catalytic Rossmann fold (RF) domain and a helical tRNA anticodon binding domain (TAB) (5). Two signature sequence motifs (KMSKS and (T/H)IGN) that participate in ATP binding typify class Ic synthetases (TrpRS, TyrRS, and PheRS). Some class I synthetases also incorporate an insertion domain (CP1) and participate in additional cellular functions such as RNA splicing, tRNA processing (6) RNA trafficking, and apoptosis (7).Although most AARSs are usually expressed from single copy genes to avoid the proliferation of synthetase mutations that could corrupt the genetic code, two copies of some synthetases are found in select bacteria and archea. In particular, Deinococcus radiodurans contains two tryptophanyl-tRNA synthetases (TrpRSs). Whereas D. radiodurans TrpRS (drTrpRS) I has 40% sequence identity to most single copy bacterial TrpRSs, drTrpRS II is more divergent, with only 28% sequence identity (8). We have recently shown that drTrpRS II interacts with D. radiodurans nitric oxide synthase (8, 9) and can charge tRNA with Trp and 4-nitrotryptophan, a specific nitration product of D. radiodurans nitric oxide synthase. drTrpRS II is induced during responses to DNA damage. It contains an unusual 30-residue extension, uncharacteristic of any other known prokaryotic TrpRSs (8) but homologous to the N terminus of the stationary phase stress-response protein, SurE (10).Extensive crystallographic work by Carter and colleagues (11-13) has established TrpRS from Bacillus stearothermophilus (bsTrpRS) as a paradigm for understanding the structural basis of enzymatic activity for class Ic AARSs. Crystal structures of bsTrpRS in three distinct conformations (open, closed, and closed pretransition state (pre-TS)) provide snapshots of important states in the catalytic cycle (12)(13)(14). An open conformation binds either ATP or Trp in a kinetic scheme described as "random bi-bi" (15, 16). Favorable binding interactions provided by the second substrate overcome the stability of the open state and result in domain closure. This high energy closed conformation (inferred in the closely related TyrRS from kinetic studies (17, 18)) engages the conserved KMSKS loop of the TAB domain with the ATP pyrophosphate group and brings the Trp carboxylate in close proximity of the ATP ␣-phosphate * This work was suppo...

Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture

“…Allostery has been reported for several aaRSs (53–56). Half of the site activity was mainly observed.…”

“…Herein, we report direct cooperative allostery for small substrate binding, as detected by ITC. Previous ITC investigations performed on other aaRSs had not detected cooperative allostery (53–56). …”

Bacterial Nitric Oxide Synthases

“…TrpRS II increases the affinity of drNOS for Arg, and in turn, drNOS perturbs fluorescent ATP analogs bound to TrpRS II. The structure of TrpRS II revealed alternations to the substrate-binding pocket, which suggested the enzyme may be able to accommodate modified forms of Trp (111,112). Indeed the enzyme was shown to charge tRNA equally well with Trp 4-nitro-Trp and 5-hydroxy-Trp (111).…”

The enzymology of nitric oxide in bacterial pathogenesis and resistance

“…TrpRS II forms a complex with D. radiodurans NOS that increases its nitration activity towards tryptophan [38]. The structure of TrpRS II revealed an unusual tryptophan-binding site that suggested that analogues of tryptophan could be recognized and potentially coupled to tRNA ( Figure 6) [40,41]. Indeed, with nearly equal specificity, TrpRS II will charge tRNA Trp with tryptophan, 4-nitrotryptophan or 5-hydroxytryptophan [40].…”