Crystal Structures of tRNA-guanine Transglycosylase (TGT) in Complex with Novel and Potent Inhibitors Unravel Pronounced Induced-fit Adaptations and Suggest Dimer Formation Upon Substrate Binding

Stengl, Bernhard; Meyer, Edgar F.; Heine, A.; Brenk, Ruth; Diederich, François; Klebe, G.

doi:10.1016/j.jmb.2007.04.008

Cited by 57 publications

(94 citation statements)

References 55 publications

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“…An N-terminal His-tag has previously been shown to have no effect on the activity of the eubacterial TGT (Todorov et al 2005). Our observation of an active dimeric human TGT is consistent with the crystallographic evidence derived from Z. mobilis TGT (Xie et al 2003;Stengl et al 2007), which indicates that the eubacterial TGT forms a homodimer. While one subunit is capable of recognizing the tRNA anticodon loop and performs the transglycosylase reaction, the other one assists in maintaining the proper orientation of bound tRNA.…”

Section: Discussionsupporting

confidence: 90%

Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

Chen

Kelly

Stachura

et al. 2010

RNA

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The eukaryotic tRNA-guanine transglycosylase (TGT) has been reported to exist as a heterodimer, in contrast to the homodimeric eubacterial TGT. While ubiquitin-specific protease 14 (USP14) has been proposed to act as a regulatory subunit of the eukaryotic TGT, the mouse TGT has recently been shown to be a queuine tRNA-ribosyltransferase 1 (QTRT1, eubacterial TGT homolog) d queuine tRNA-ribosyltransferase domain-containing 1 (QTRTD1) heterodimer. We find that human QTRTD1 (hQTRTD1) co-purifies with polyhistidine-tagged human QTRT1 (ht-hQTRT1) via Ni 2+ affinity chromatography. Cross-linking experiments, mass spectrometry, and size exclusion chromatography results are consistent with the two proteins existing as a heterodimer. We have not been able to observe co-purification and/or association between hQTRT1 and USP14 when coexpressed in Escherichia coli. More importantly, under our experimental conditions, the transglycosylase activity of hQTRT1 is only observed when hQTRT1 and hQTRTD1 have been co-expressed and co-purified. Kinetic characterization of the human TGT (hQTRT1 d hQTRTD1) using human tRNA Tyr and guanine shows catalytic efficiency (k cat /K M ) similar to that of the E. coli TGT. Furthermore, site-directed mutagenesis confirms that the hQTRT1 subunit is responsible for the transglycosylase activity. Taken together, these results indicate that the human TGT is composed of a catalytic subunit, hQTRT1, and hQTRTD1, not USP14. hQTRTD1 has been implicated as the salvage enzyme that generates free queuine from QMP. Work is ongoing in our laboratory to confirm this activity.

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Section: Discussionsupporting

confidence: 90%

Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

Chen

Kelly

Stachura

et al. 2010

RNA

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“…Such bases are introduced by TGT. [6] It exchanges the nucleobase guanine for a modified base in the wobble position 34 of tRNA His,Asn,Asp,Tyr . [9,10] Our drug development strategy is therefore to inhibit the base exchange reaction of TGT to decrease the virulence of Shigella bacteria.…”

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confidence: 99%

How to Replace the Residual Solvation Shell of Polar Active Site Residues to Achieve Nanomolar Inhibition of tRNA‐Guanine Transglycosylase

et al. 2009

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In a computational and structural study, we investigated a series of 4-substituted lin-benzoguanines that are potent inhibitors of tRNA-guanine transglycosylase (TGT), a putative target for the treatment of shigellosis. At first glance, it appears self-evident that the placement of a positively charged ligand functional group between the carboxylate groups of two adjacent aspartate residues in the glycosylase catalytic center leads to enhanced ligand binding. The concomitant displacement of water molecules that partially solvate the aspartates prior to ligand binding appears to result as a consequence of this. However, the case study presented herein shows that this premise is much too superficial. Placement of a likely positively charged amino group at such a pivotal position, interfering with the residual water solvation shell, is at best cost-neutral compared with the unsubstituted parent ligand not conflicting with the residual water shell. A ligand that orients a hydroxy group in this position shows even decreased binding. Based on the cost-neutral placement of the amino functionality, hydrophobic side chains can now be further attached to fill, with increasing potency, a small hydrophobic pocket remote to the aspartates. Any attempts to cross the pivotal position between both aspartates with nonpolar scaffolds reveals only decreased binding, even though the waters of the residual solvation shell are successfully repelled. This surprising observation fostered a detailed analysis of the role of water molecules involved in the residual solvation of polar active site residues. Their geometry and putative replacement in the binding pocket of TGT has been studied by a comparative database analysis, computational active site mapping, and a series of crystal structure analyses. Furthermore, conformational preferences of attached hydrophobic moieties explain their contribution to a gradual increase in binding affinity.

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“…[7,8] Some of these ligands afforded inhibitory constants K i down to the single-digit nanomolar range. [7,8] Here, we report a new series of highly active ligands featuring a substituent in position 4 to address the hydrophobic surface depression shaped by Val45 and Leu68 near the ribose-34 site without paying, as with previous ligands, [5,6] a large energetic penalty for replacing a consensus water cluster solvating the two catalytic Asp side chains (Asp102 and Asp280) of the enzyme.…”

Section: Introductionmentioning

confidence: 97%

“…We have previously introduced 6-amino-imidazoA C H T U N G T R E N N U N G [4,5-g]quinazolin-8(7H)-one (lin-benzoguanine) [4] as a promising scaffold for the design of TGT inhibitors. [5,6] We subsequently demonstrated the large potential of this heterotricyclic scaffold with a series of inhibitors featuring amino substituents in position 2, which reach into the spacious ribose-33 pocket (for the numbering see Table 1). [7,8] Some of these ligands afforded inhibitory constants K i down to the single-digit nanomolar range.…”

Section: Introductionmentioning

confidence: 99%

“…[19] A superposition of the crystal structure of inhibitor 2 in the active site with this consensus water cluster ( Figure 1) reveals an explanation for the relatively poor affinities of inhibitors 2-4: The water cluster is essential for the solvation of the negatively charged residues of Asp102 and Asp280. [6,19] Upon binding of an appropriately substituted inhibitor, the substituent attached to address the hydrophobic surface depression to accommodate ribose-34 displaces the water molecules to varying extent, leaving the aspartates "naked". This results in Coulomb repulsion of the two most likely negatively charged residues as well as unfavorable CO 2 À ···CH 2 interactions with the apolar substituent, which cause the side chain to distribute over two conformations.…”

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confidence: 99%

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High‐Affinity Inhibitors of tRNA‐Guanine Transglycosylase Replacing the Function of a Structural Water Cluster

Köhler

Ritschel

Schweizer

et al. 2009

Chemistry A European J

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The tRNA‐modifying enzyme tRNA–guanine transglycosylase (TGT) is essential for the pathogenic mechanism of Shigella flexneri, the causing agent of the bacterial diarrheal disease shigellosis. Herein, the synthesis of a new class of rationally designed 6‐amino‐imidazo[4,5‐g]quinazolin‐8(7H)‐one‐ (lin‐benzoguanine) based inhibitors of TGT are reported. In order to accommodate a small hydrophobic crevice opening near the binding site of ribose‐34, 2‐aminoethyl substituents were introduced in position 4 of the heterocyclic scaffold. For this purpose, a synthetic sequence consisting of iodination, Suzuki cross‐coupling, hydroboration, Mitsunobu reaction, and Gabriel synthesis was employed, furnishing a primary amine that served as a common intermediate for the preparation of a series of derivatives. The resulting ligands displayed very low inhibition constants, down to Ki=2 nM. Substantial additional inhibitory potency is gained by interaction of terminal lipophilic groups attached to the substituent at position 4 with the hydrophobic crevice shaped by Val45 and Leu68. At the same time, the secondary ammonium center in the substituent displaces a cluster of water molecules, solvating the catalytic residues Asp102 and Asp280, without loss in binding affinity. In addition, a synthetic intermediate with an unusual 3,6,7,8,9,10‐hexahydroimidazo[4,5‐g][1,3]benzodiazepine core, as confirmed by X‐ray analysis, is reported.

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Crystal Structures of tRNA-guanine Transglycosylase (TGT) in Complex with Novel and Potent Inhibitors Unravel Pronounced Induced-fit Adaptations and Suggest Dimer Formation Upon Substrate Binding

Cited by 57 publications

References 55 publications

Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

How to Replace the Residual Solvation Shell of Polar Active Site Residues to Achieve Nanomolar Inhibition of tRNA‐Guanine Transglycosylase

High‐Affinity Inhibitors of tRNA‐Guanine Transglycosylase Replacing the Function of a Structural Water Cluster

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