Crystallographic data collection and refinement statistics – Tgt(Cys158Val/Val233Gly), “WT”.

<p>Crystallographic data collection and refinement statistics – Tgt(Cys158Val/Val233Gly), “WT”.</p

Substrate base binding pocket of <i>Z.</i> <i>mobilis</i> Tgt and modelled human Tgt.<b> </b>

<p>A) Detail of <i>Z. mobilis</i> Tgt·preQ₁ complex crystal structure (PDB-code: <b><u>1p0e</u></b>) showing the active site with the bound substrate in stick representation. Carbon atoms of protein residues are coloured in green, those of preQ₁ in orange. B) Homology model of human Tgt created with the <i>Z. mobilis</i> Tgt crystal structure as a template. The close up shows active site residues (carbon atoms in grey) superimposed with preQ₁ (carbon atoms in orange) as present in <b><u>1p0e</u></b>. The coordinates of the homology model are provided within the Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064240#pone.0064240.s001" target="_blank">Coordinates S1</a>).</p

Launching Spiking Ligands into a Protein–Protein Interface: A Promising Strategy To Destabilize and Break Interface Formation in a tRNA Modifying Enzyme

Apart from competitive active-site inhibition of protein function, perturbance of protein–protein interactions by small molecules in oligodomain enzymes opens new perspectives for innovative therapeutics. tRNA–guanine transglycosylase (TGT), a potential target to treat shigellosis, is active only as the homodimer. Consequently, disruption of the dimer interface by small molecules provides a novel inhibition mode. A special feature of this enzyme is the short distance between active site and rim of the dimer interface. This suggests design of expanded active-site inhibitors decorated with rigid, needle-type substituents to spike into potential hot spots of the interaction interface. Ligands with attached ethinyl-type substituents have been synthesized and characterized by <i>K</i>_d measurements, crystallography, noncovalent mass spectrometry, and computer simulations. In contrast to previously determined crystal structures with nonextended active-site inhibitors, a well-defined loop-helix motif, involved in several contacts across the dimer interface, falls apart and suggests enhanced flexibility once the spiking ligands are bound. Mass spectrometry indicates significant destabilization but not full disruption of the complexed TGT homodimer in solution. As directed interactions of the loop-helix motif obviously do not determine dimer stability, a structurally conserved hydrophobic patch composed of several aromatic amino acids is suggested as interaction hot spot. The residues of this patch reside on a structurally highly conserved helix-turn-helix motif, which remains unaffected by the bound spiking ligands. Nevertheless, it is shielded from solvent access by the loop-helix motif that becomes perturbed upon binding of the spiking ligands, which serves as a possible explanation for reduced interface stability

Superimposition of preQ₁-bound Tgt(Cys158Val) with its apo-, its guanine- and its queuine-bound form.

<p>Carbon atoms of the Tgt(Cys158Val)·preQ₁ complex are coloured pink in all sub-figures. (A) Superimposition (based on c_α) of preQ₁-bound Tgt(Cys158Val) and apo-Tgt(Cys158Val). Carbon atoms of apo-Tgt(Cys158Val) are coloured blue. Binding of preQ₁ to Tgt(Cys158Val) provokes a shift of Val158 towards the ligand leading to the displacement of two water molecules (W1 and W2; shown as red spheres) which are present within this region in apo-Tgt(Cys158Val) and absent in the complex structure. In addition, the side chain of the proximate Thr159 rotates by about 90°. Exactly the same structural changes upon binding of preQ₁ are observed for Tgt(Cys158Val/Val233Gly). (B) Superimposition (based on c_α) of preQ₁-bound Tgt(Cys158Val) and guanine-bound Tgt(Cys158Val). Carbon atoms of the Tgt(Cys158Val)·guanine complex are coloured yellow. In the Tgt(Cys158Val)·guanine complex, the side chain of Val158 becomes largely disordered. The Thr159 side chain adopts a similar conformation as observed in the apo-structure. (C) Superimposition (based on c_α) of preQ₁-bound Tgt(Cys158Val) and queuine-bound Tgt(Cys158Val). Carbon atoms of the Tgt(Cys158Val)·queuine complex are shown in grey. Binding of queuine obviously leads to disordering of the Val158 side chain as no electron density attributable to this isopropyl moiety is present in the electron density map of the refined Tgt(Cys158Val)·queuine complex structure. Also upon binding of queuine no conformational change of the Thr159 side chain is observed. It adopts a similar conformation as in the apo- and guanine-bound structures.</p

Trapping experiments performed with Tgt/tRNA mixtures in presence of queuine or 2,6-diamino-3<i>H</i>-quinazolin-4-one.

<p>A) Chemical structure of 2,6-diamino-3<i>H</i>-quinazolin-4-one (DAQ), an uncompetitive inhibitor of Tgt. B) SDS-PAGE analysis of reaction mixtures of Tgt or mutated variants thereof and tRNA^Tyr under conditions indicated. SM, size marker; q, queuine. While DAQ causes retarded Tgt bands by stabilising the covalent Tgt·tRNA intermediate, queuine lacks this ability for most of the investigated Tgt variants. Solely in case of Tgt(Cys159Val/Val233Gly) a faint retarded band is visible indicating that queuine may to some extent be able to bind to the guanine 34/preQ₁ subpocket of the covalent enzyme·tRNA complex.</p

Crystallographic data collection and refinement statistics - Tgt(Cys158Val).

<p>Crystallographic data collection and refinement statistics - Tgt(Cys158Val).</p

Crystal structures of Tgt variants in complex with preQ₁ or queuine: surface representation of substrate pockets.

<p>The solvent accessible surfaces of active sites from <i>Z. mobilis</i> Tgt variants are shown in bright yellow. The respective Tgt variant plus the bound ligand (shown in stick representation) are indicated in each sub-figure. Also amino acid residues at positions 158 and 233 are shown in stick representation. Carbon atoms of original amino acids and of the bound ligand are coloured green, those of mutated amino acids magenta. As the electron density assignable to the dihydroxy-cyclopentenyl moiety of queuine is poorly defined in all structures containing this ligand the coordinates of this moiety are not present in the respective structures deposited with the Protein Data Base. Accordingly, the conformations of the dihydroxy-cyclopentenyl shown in (D), (F) and (H) are tentative. To indicate this fact, the carbon atoms of this moiety are shown in grey. Selected water molecules are shown as red spheres. 2|F_o|-|F_c| (at σ 1.0) electron density is shown for the bound ligand and water molecules. 2|F_o|-|F_c| electron density contoured at a σ level of 1.0 is coloured blue, |F_o|-|F_c| electron density contoured at a σ level of 2.5 is coloured magenta. An overview of the crystal structures analysed in this study including resolutions and PDB codes is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064240#pone.0064240.s004" target="_blank">Table S1</a>.</p

Kinetic parameters for “wild type” Tgt and mutated Tgt variants.^*

<p>Kinetic parameters for “wild type” Tgt and mutated Tgt variants.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064240#nt101" target="_blank">*</a>}</p

Crystal structures of Tgt variants in complex with preQ₁ or queuine: stick representation of substrate pockets.

<p>The respective Tgt variant plus the bound ligand are indicated in each sub-figure. Carbon atoms of original amino acids are coloured green, those of mutated amino acids as well as of the bound ligand orange. As the electron density assignable to the dihydroxy-cyclopentenyl moiety of queuine is poorly defined in all structures containing this ligand the coordinates of this moiety are not present in the respective structures deposited with the Protein Data Base. Accordingly, the conformations of the dihydroxy-cyclopentenyl shown in (D), (F) and (H) are tentative. To indicate this fact, the carbon atoms of this moiety are shown in grey. Selected water molecules are shown as red spheres. Electron density is shown for amino acid residues at positions 158 and 233 as well as for the ligand and for water molecules. The 2|F_o|-|F_c| electron density map contoured at a σ level of 1.0 is coloured blue. The green density represents an |F_o|-|F_c| omit map contoured at 2.5 σ. An overview of the crystal structures analysed in this study including nominal resolutions and PDB codes is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064240#pone.0064240.s004" target="_blank">Table S1</a>.</p

Assumed catalytic mechanism of bacterial Tgt.

<p>(A and B) The glycosidic bond of guanosine 34 is cleaved <i>via</i> nucleophilic attack by the Asp280 carboxylate resulting in the formation of a covalent Tgt⋅tRNA intermediate. (C and D) Guanine is replaced by preQ₁ which is incorporated into the tRNA <i>via</i> nucleophilic attack of the ribose 34 anomeric carbon by <i>N</i>9 of preQ₁. Notably the replacement of guanine by preQ₁ in the binding pocket of Tgt induces a flip of the Leu231/Ala232 peptide bond. The formation of a hydroxide and an oxonium ion as byproducts of the reaction is assumed to be responsible for its irreversibility as mutual neutralisation will efficiently detract these ions from equilibrium. <i>H</i>-bonds are indicated by dashed lines.</p