Computational protocol: Investigation of Specificity Determinants in Bacterial tRNA-Guanine Transglycosylase Reveals Queuine, the Substrate of Its Eucaryotic Counterpart, as Inhibitor

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[…] The crystal structure of Z. mobilis Tgt (PDB code 1r5y chain A) was used as a template to create a model of the human Tgt catalytic subunit (UniProtKB/Swiss-Prot accession code Q9BXR0). Using ClustalW 1.83 the corresponding sequences aligned with 42% identity. Based on the alignment, ten homology models for human Tgt were calculated with MODELLER 6a . As no significant differences were apparent, the model with the lowest MODELLER target function value (11,429.19) was selected as representative. The coordinates of the homology model are provided within the Supporting Information (). [...] Crystals of the investigated Tgt variants suitable for ligand soaking were grown via the hanging-drop vapour diffusion method. Droplets were prepared by mixing 1 µL of concentrated protein solution (12 to 15 mg·mL−1 in 2 mol·L−1 NaCl, 10 mmol·L−1 Tris/HCl pH 7.8, 1 mmol·L−1 EDTA, 1 mmol·L−1 DTT) with 1 µL reservoir solution (11% (w/v) PEG 8,000, 100 mmol·L−1 Tris/HCl, pH 8.5, 1 mmol·L−1 DTT, 10% (v/v) DMSO). Crystals grew within one week at 18°C in the presence of 1.0 mL of reservoir solution.For cocrystallisation of Tgt(Cys158Val) with guanine, guanine·HCl was dissolved in DMSO and added to the crystallisation droplet to a final concentration of 5 mmol·L−1. As guanine is, under the above-named conditions, not soluble up to this concentration a substantial portion of the compound precipitated. Crystals grew within the guanine precipitate in two weeks. To allow cocrystallisation of the Tgt variants with Q the compound was dissolved in DMSO and added to the droplet to a final concentration of 20 mmol·L−1. Crystals of the Tgt variants complexed to preQ1 were produced by a soaking procedure. The compound was dissolved in DMSO and added to 2 µL of reservoir solution to a final concentration of 10 to 20 mmol·L−1. Finally, a single pre-grown Tgt crystal was transferred into the droplet which was sealed against 1.0 mL of reservoir solution and soaked for 30 min.For data collection, crystals were cryoprotected using glycerol. The glycerol and PEG 8,000 concentrations of the reservoir buffers were increased stepwise by transferring the crystals to six different 2 µL cryodroplets each with 30-min incubation times while sealed against 1.0 mL of the same solution (glycerol concentrations (v/v): 5%→10%→15%→20%→25%→30%; and PEG 8,000 concentrations (w/v): 5.0%→6.3%→7.5%→8.0%→8.8%→9.8%, respectively). These droplets also contained the ligands at the same concentrations as the soaking and cocrystallisation solutions. The cryo-soaked crystals were flash-frozen in liquid nitrogen. Data sets (3bld, 4dg0, 4e2v, 4hvx, 4h7z, 4h6e, 4hsh, 4hqv, 4gcx and “WT”-Tgt·queuine) were collected at cryo conditions (−173°C≡100 K) at the BESSY II (Helmholz-Zentrum, Berlin, Germany) beamlines BL-14.1 (λ = 0.91841 Å) and BL-14.2 (λ = 0.91841 Å) and with CuKα radiation (λ = 1.5418 Å) using a Rigaku RU-300 rotating-anode generator at 50 kV and 90 mA equipped with Xenocs focussing optics and an R-AXIS IV detector. All crystals tested exhibited monoclinic symmetry in space group C2 containing one monomer per asymmetric unit with Matthews coefficients of 2.3–2.4. Data processing and scaling was performed using the HKL2000 package except for structure 4h7z which was processed using iMOSFLM 1.0.6 and SCALA . For all refined structures unit cell dimensions for the crystals, data collection and processing statistics are given in , and .Coordinates of the apo-Tgt crystal structure (PDB-code: 1pud in case of the structures 3bl3, 3bld, 3blo) or rather coordinates of Tgt in complex with 2-[(thiophen-2-ylmethyl)amino]-1,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one (PDB-code: 3gev in case of structures 4dg0, 4e2v, 4hvx, 4h7z, 4h6e, 4hsh, 4hqv, 4gcx and “WT”-Tgt·queuine) were slightly modified (deletion of water and ligand as well as coordinates at the sites of mutation) before applying for initial rigid-body refinement of the protein molecule followed by repeated cycles of conjugate gradient energy minimisation, simulated annealing and B-factor refinement using the CNS program package . Refinement at the later stages was performed with SHELXL for structures 3bl3, 3bld, and 3blo. Here, up to 50 cycles of conjugate gradient minimisation were performed with default restraints on bonding geometry and B-values: 5% of all data were used for Rfree calculation. For the structures 4e2v, 4dg0, 4h7z, 4h6e, 4hqv, 4hsh, 4hqv, 4gcx and “WT”-Tgt·queuine refinement was performed with PHENIX 1.8-1069 with 5% to 10% of all data being used for Rfree calculation and followed by repeated cycles of maximum likehood energy minimization. For the structures 4h7z, 4hvx, 4hqv, 4hsh an additional optimization of the weights between X-ray target and stereochemistry as well as between ADP restraints was performed by PHENIX 1.8-1069 . Amino acid side chains were fitted to σA-weighted 2|Fo| - |Fc| and |Fo| - |Fc| electron density maps using Coot . Ligands were generated using SYBYL 8.0 (Tripos International: 1699 South Hanley Rd., St. Louis, Missouri, 63144, USA). Afterwards, geometric restraints of the ligands were calculated by Monomer Library Sketcher (ccp4) . For structures 4e2v, 4dg0, 4h7z, 4h6e, 4hqv, 4hsh, 4hqv, 4gcx and “WT”-Tgt·queuine water was located using the refinement settings “update waters” implemented in the program PHENIX 1.8-1068 . After further refinement cycles water was checked visually. Glycerol molecules and ligands were located in the difference electron density and added to the model for further refinement cycles. The temperature factors (individual B-factors) for structures 4e2v, 4gcx, 4gd0 and 3bld were anisotropically refined, whereas for structures 4hvx, 4h7z, 4hsh, 4hqv and 4h6e and “WT”-Tgt·queuine TLS refinement was applied. The definition of the TLS groups was done with the TLSMD server . Restraints were applied to bond lengths and angles, planarity of aromatic rings, and Van-der-Waals contacts. Multiple side-chain conformations were built in case an appropriate electron density was observed and maintained during refinement and if the minor populated side chain showed at least 20% occupancy. All final models were validated using PROCHECK . Analysis of temperature factors was done with Moleman . [...] Alignment of structures based on cα with similar or identical sequences was performed with the alignment function implemented in Pymol ( Figures were prepared using ChemDraw Std 12.0 (PerkinElmer, Massachusetts, USA) and Pymol. […]

Pipeline specifications

Software tools Clustal W, MODELLER, SHELX, iMosflm, CCP4, CNS, PHENIX, Coot, PROCHECK, PyMOL, ChemDraw
Databases UniProt UniProtKB
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Caenorhabditis elegans, Bacteria, Homo sapiens, Zymomonas mobilis
Chemicals Glycine, Guanine, Tyrosine