Fig 1.
Overview of the active site, its different sub-pockets (yellow, orange, magenta), and part of the second monomer.
Co-crystal structure of TGT with inhibitor 5 (TGT∙5co). The protein is shown as transparent solvent accessible surface and the loop-helix motif as cartoon. Color code: C green, O red, N blue. 5 and selected residues are displayed as sticks. For the sake of clarity, residues Tyr106 to Leu111 are not shown. Val45, Leu68 and Val282 form the hydrophobic floor of the ribose-34/phosphate-35 pocket (orange). Selected portions of the second monomer of the homodimer are shown as blue cartoon. Blue dashed lines indicate H-bonds from 5 with the protein (2.7–2.9 Å). These characteristics apply to all following figures unless otherwise stated. N5 of the lin-benzoguanine scaffold was shown to be protonated upon binding to TGT allowing Asp102 to form two H-bonds to the ligand [29].
Fig 2.
Overview of studied TGT inhibitors, inhibition constants (Ki), and crystal structures.
Fig 3.
Binding modes and structural arrangements of 2, 3 and 5–8.
Color coding for carbons in parenthesis. Comparison of a) TGT∙2co (green) and TGT∙3co (yellow), b) TGT∙3soak (conformation B cyan and conformation A gray) and TGT∙3co (yellow), c) TGT∙5soak (gray) and TGT∙5co (green), d) TGT∙6soak (gray) and TGT∙6co (yellow), e) TGT∙7soak (TGT1 gray; TGT2 blue) and TGT∙7co (yellow), f) TGT∙8soak (conformation A: cyan and conformation B gray) and TGT∙8co (yellow). Selected water molecules are shown as spheres and colored as the corresponding complex. For the sake of clarity, residues of the guanine-34/preQ1 binding site that interact with the lin-benzoguanine scaffold are not shown. Solvent accessible surface and selected residues as sticks of the second monomer of the homodimer are colored gray. Position of missing Thr47 in TGT∙3soak is indicated as gray dashed lines. Black arrows indicate positional differences between the crystal structures of different crystallization protocols. Occupancies for the different side chains are displayed using the corresponding color of the ligand. Inhibitors 3, 6, 7 and 8 displace the βα1-loop in the co-crystal structures. Colored dashed lines indicate H-bonds from selected water molecules (2.4–3.5 Å).
Table 1.
Data collection and refinement statistics of X-ray crystal structures.
Fig 4.
Binding modes and structural rearrangements of 9.
a) TGT∙9soak (conformation A gray and conformation B cyan) and TGT∙9co (yellow), b) View from an alternative direction including the second monomer of TGT∙9co in light blue and with gray solvent accessible surface. Black arrows indicate positional differences between the crystal structures of different crystallization protocols. Inhibitor 9 displaces the β1α1-loop in the co-crystal structure and triggers the formation of a new twisted dimer (see Figure 4d). Yellow dashed lines indicate H-bonds from selected water molecules (2.7–3.4 Å).
Fig 5.
Comparison of alternative TGT dimer interfaces and oligomeric states.
Dimer interface areas calculated using PDBePISA49 are highlighted by red surface patches and stick representation. a) apo-TGT dimer interface area of monomer 1: 1619 Å2 (PDB entry: 1P0D [18]). b) twisted dimer interface of monomer 1: 1644 Å2 (found in TGT∙9co) c) oligomeric state of apo-TGT dimer (monomer 1 red, monomer 2 blue) d) oligomeric state of twisted dimer (monomer 1 red, monomer 2 blue).
Fig 6.
Polar interactions formed in a) apo-TGT (PDB entry: 1P0D [18]) and b) twisted dimer found in TGT∙9co.
Residues that form H-bonds or salt bridges across the interface only in apo-TGT (yellow), in both structures (green), and in the twisted dimer (red) are indicated. Contacts were determined by CONTACTSYM [47, 48]. Due to interface symmetry, all displayed interactions occur twice but are depicted only once for the sake of clarity. Only H-bonds (blue dashed lines) and salt bridges (red dashed lines) with distances between 2.6 and 3.7 Å are depicted.
Fig 7.
Illustration of the aromatic hot spot regions.
H-bonds and salt bridges are displayed as black dashed lines. a) Arrangement of the aromatic hot spot region formed in apo-TGT (PDB entry: 1P0D[18]). The β1α1-loop of the loop-helix motif shields this region from water access and can be perturbed by inhibitor binding (monomer 1 red, monomer 2 blue, space group C2). b) New arrangement of the hot spot region in the twisted dimer found in TGT∙9co (monomer 1 red, monomer 2 blue, space group P21).
Fig 8.
Native MS characterization of TGT dimer disturbing potential from each ligand.
Relative quantification of TGT monomers and dimers signals was performed in the absence (TGT 2.5μM) or presence of each ligand (TGT:ligand 1:10 mixture). TGT monomer ratios (%) deduced from each ligand mixture (S7 Fig; S1 Table) were normalized with respect to the apo-condition, enabling to rank inhibitors according to their destabilization factor. Corresponding means and standard deviations (black error bars) are derived from triplicate measurements.
Fig 9.
Plot of ΔH0obs against the buffer ΔH0ion and thermodynamic profiles.
a) Measured heat signal (ΔH0obs) is plotted against the ionization enthalpy of applied buffers (HEPES, Tricin, TRIS) to extract the corrected enthalpy of binding (ΔH0bind). Inhibitor 1 blue, 2 black, and 5 green. b) Thermodynamic parameters ΔG0 (blue) and corrected values for ΔH0bind (green) and -TΔS0 (red) in kJ∙mol-1.
Table 2.
Inhibition constants and thermodynamic data at the applied buffers.
Fig 10.
Alignment of TGT∙RNA (PDB entry: 1Q2R [14]) and twisted dimer found in TGT∙9co.
The protein is represented as solvent accessible surface and cartoon with the following color code: twisted dimer: Monomer 1 red, Monomer 2 yellow. TGT∙RNA: Monomer 1 adopts a very similar orientation in space as in the twisted dimer (for the sake of clarity not shown in the figure). Monomer 2 blue. RNA: backbone orange and nucleosides green sticks.