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

Abstract

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 Kd 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.

Soaking suggests “alternative facts”: Only co-crystallization discloses major ligand-induced interface rearrangements of a homodimeric tRNA-binding protein indicating a novel mode-of-inhibition

“…In a former study, we had shown that the structure of TGT co-crystallized with 4 (TGT∙ 4 co ) reveals a binding mode which is significantly different from that seen in the structure obtained after soaking 4 into an apo-TGT crystal (TGT∙ 4 soak ) (Fig 2 and S2 Fig) [22]. Similar to TGT∙ 3 co , the adopted binding mode in TGT∙ 4 co leads to a pronounced rearrangement of the loop-helix motif accompanied by conspicuous changes of unit cell parameters (reduction of the a -axis and shrinking of the β-angle).…”

“…This technique enables to monitor the oligomerization state of TGT at equilibrium under non-denaturing conditions, in the absence or presence of saturating ligand concentrations [15, 22, 24, 25]. Previously, we found that relative proportions of TGT monomer were strongly dependent on experimental (protein concentration, batch, conditioning) and instrumental settings (cone voltage, backing pressure).…”

“…In previous studies, we had characterized a number of TGT inhibitors, such as 1 – 9 in Fig 2, which were all based on a lin -benzoguanine scaffold endowed with various substituents addressing the adjacent recognition pockets [15, 19–22]. While the lin -benzoguanine scaffold invariably occupies the guanine-34/preQ 1 binding site, its substituents reach into adjacent sub-pockets thereby leading to a broad range of TGT affinities.…”

“…In addition, helix α 1 is no longer crystallographically resolved. These structural elements, hereinafter referred to as loop-helix motif , usually shield a cluster of aromatic residues within the TGT homodimer interface from water access [22, 24, 25]. While in our previous work we focused on the interactions of ligands 2 and 3 with active-site residues of bacterial TGT [23], here we investigate their distinct effects on dimer interface architecture caused by the mere presence or absence of a sole methyl group at the furanoside 3´-OH function.…”

“…This ligand had been designed to launch a rigid 3-pyridyl-ethynyl needle in the homodimer interface with the intention to destabilize it [22]. Remarkably, the named conformational changes of TGT as well as changes in unit cell parameters did not appear when 4 was soaked into a pre-existing apo-TGT crystal (S2 Fig).…”