Molecular dynamics simulations of LysRS: An asymmetric state

Hughes, Samantha Jane; Tanner, Julian A.; Miller, Andrew D.; Gould, Ian R.

doi:10.1002/prot.20609

Cited by 12 publications

(8 citation statements)

References 21 publications

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“…In addition, GTP was found to be a significantly poorer inhibitor of Hint-AMP formation than ATP. In support of our findings, recent modeling studies attempting to develop a molecular rationale for the substrate specificity of ecLysU have concluded that the enzyme has a greater propensity to bind ATP in the necessary productive conformation than GTP (44).…”

Section: Discussionsupporting

confidence: 85%

Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins

Chou

Wagner

2007

Journal of Biological Chemistry

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Histidine triad nucleotide binding proteins (Hints) are the most ancient members of the histidine triad protein superfamily of nucleotidyltransferases and hydrolyases. Protein-protein interaction studies have found that complexes of the transcription factors MITF or USF2 and lysyl-tRNA synthetase (LysRS) are associated with human Hint1. Therefore, we hypothesized that lysyl-AMP or the LysRS⅐lysyl-AMP may be a native substrate for Hints. To explore the biochemical relationship between Hint1 and LysRS, a series of catalytic radiolabeling, mutagenesis, and kinetic experiments was conducted with purified LysRSs and Hints from human and Escherichia coli. Histidine triad nucleotide binding protein (Hint) 2 belongs to a histidine triad (HIT) superfamily that has a characteristic C-terminal active site motif, HXHXHXX, where X is a hydrophobic residue (1). HIT enzymes are a ubiquitous superfamily consisting primarily of nucleoside phosphoramidases, dinucleotide hydrolyases, and nucleotidyltransferases (1). Five distinct branches of HIT superfamily have been recently classified by a phylogenetic study of HIT proteins (2, 3). Recently, Hint1 homologs isolated from rabbit (4), human (5), chicken (6), yeast (4), and Escherichia coli (5) have been shown to be purine nucleoside phosphoramidases. Human Hint1 has been shown to associate with, and possibly regulate several transcription factors such as TFIIH (7), MITF (8, 9), and USF2 (10). Additionally, Hint1 knock out mice have been shown to have an increased susceptibility to the induction of ovarian and mammary tumors by the carcinogen dimethylbenzanthracene and to spontaneous tumors (11). Up-regulation of Hint1 and the significantly reduced in vivo tumorigenicity of 5-aza-dC-treated non-smallcell lung cancer cell line NCI-H522 suggested that hHint1 might be a tumor suppressor (12). Recently, Weiske and Huber (13) reported that Hint1 triggers apoptosis independent of its phosphoramidase activity (13). Human Hint2, which is 61% identical to Hint1, has recently been shown to be a mitochondrial apoptotic sensitizer that is down-regulated in hepatocellular carcinomas (14). Although Hints are efficient hydrolases of purine nucleoside phosphoramidates, cellular function and biochemical relevance of the enzymatic phosphoramidase activity has not been determined.The Fhit (fragile histidine triad) branch of the HIT superfamily is only found in eukaryotes. Like Hint, Fhit is also a homodimer with tumor suppressor activity (15). Human Fhit is a diadenosine P 1 ,P 3 -triphosphate (Ap 3 A) hydrolyase as well as phosphoramidase (16,17). The precise mechanism of action by which it affects tumor development is not well understood, although site-directed mutagenesis studies have indicated that the Fhit tumor suppressor function is likely not dependent on its Ap 3 A hydrolase activity (18). In contrast to the first two branches, the galactose-1-phosphate uridylyltransferase branch has been shown to be the second enzyme in the Leloir pathway necessary for galactose utilization (19,20). Although...

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Section: Discussionsupporting

confidence: 85%

Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins

Chou

Wagner

2007

Journal of Biological Chemistry

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“…A number of oligomeric aaRS have previously been shown to exhibit activity of half of the sites despite containing multiple, seemingly chemically equivalent active and tRNA-binding sites (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)30). The mode of tRNA binding described herein argues that SepSecS may follow the sequential model of allosteric regulation (49).…”

Section: Discussionmentioning

confidence: 83%

“…The TyrRS dimer binds one tRNA Tyr (15) and catalyzes formation of 1 mol of tyrosyl adenylate (16 -20), and tetrameric PheRS (15,21) and SepRS (22) employ two of their four catalytic and tRNA-binding sites at a time. Likewise, class II enzymes, bacterial LysRS-II (23,24), AspRS (25), and HisRS (26) and the archaeal and eukaryotic ProRS (27,28) behave according to the "half of the sites" model. The situation is less clear in the case of PylRS and SerRS.…”

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confidence: 99%

Structural Asymmetry of the Terminal Catalytic Complex in Selenocysteine Synthesis

French

Gupta

Copeland

et al. 2014

Journal of Biological Chemistry

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Background: SepSecS catalyzes the terminal step of selenocysteine synthesis in a reaction that requires tRNA Sec . Results: SepSecS simultaneously binds up to two tRNAs in a cross-dimer fashion. Conclusion:The SepSecS-tRNA Sec complex is asymmetric: one dimer is a noncatalytic platform that binds tRNAs, whereas the other one catalyzes the reaction. Significance: The study proposes allosteric regulation of selenocysteine and selenoprotein synthesis.

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“…In site 2, R269 appears to act as a lid to close the binding pocket; in site 1, this lid has been displaced leaving the pocket open. Other active site/first shell amino acid residues of potential importance to Ap 4 A/Ap 3 A formation can be seen by inspection of the molecular dynamics simulation and X‐ray crystal structures of LysU with bound substrates (Fig. A).…”

Section: Introductionmentioning

confidence: 99%

“…LysU is homodimeric with one active site associated with each monomeric polypeptide. Previous order-of-addition experiments [16,17] show that LysU is initially loaded at each site with Mg 2+ and L-lysine, giving a LysU:6Mg 2+ : L-lysine 2 intermediate state with significant conformational distortion. Thereafter, ATP binds to a single active site (designated 2) developing a structural asymmetry culminating in distortions in the motif 2 loop at site 1.…”

Section: Introductionmentioning

confidence: 99%

Multiple catalytic activities of Escherichia coli lysyl‐tRNA synthetase (LysU) are dissected by site‐directed mutagenesis

et al. 2012

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The heat-inducible lysyl-tRNA synthetase from Escherichia coli (LysU; EC 6.1.1.6) converts ATP to diadenosine tri-and tetraphosphates (Ap 3 A/ Ap 4 A) in the presence of L-lysine/Mg 2+ /Zn 2+ . To understand LysU in more detail, 26 mutants were prepared: six of E264, four of R269 and sixteen mutants by alanine-scanning of the inner shell/motif 2 loop. In the presence of glycerol and absence of exogenously added Zn 2+ /L-lysine, we unexpectedly found that E264K catalysed the production of glycerol-3-phosphate, powered by ATP turnover to ADP. E264Q and E264N are also capable of this activity, but all three show little formation of Ap 4 A/Ap 3 A under normal conditions (additional Zn 2+ /L-lysine/Mg 2+ ). By contrast, wild-type LysU has a weaker glycerol kinase-like capability in the absence of Zn 2+ and is dominated by Ap 4 A/Ap 3 A synthesis in its presence. Kinetic and isothermal titration calorimetry results suggest that E264 is a crucial residue for Zn 2+ promotion of Ap 4 A/Ap 3 A synthesis. This is consistent with the hypothesis that E264 provides an anchor point for a Zn 2+ ion complexed to the active site, with simultaneous coordination to the enzyme bound lysyl-adenylate intermediate and secondary substrate ATP/ADP. The glycerol kinase-like activity is uncovered on disruption of this specific coordination.

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Molecular dynamics simulations of LysRS: An asymmetric state

Cited by 12 publications

References 21 publications

Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins

Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins

Structural Asymmetry of the Terminal Catalytic Complex in Selenocysteine Synthesis

Multiple catalytic activities of Escherichia coli lysyl‐tRNA synthetase (LysU) are dissected by site‐directed mutagenesis

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