title: 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.
authors.affiliation: Institut für Pharmazeutische Chemie, Philipps-Universität Marburg , Marbacher Weg 6, 35032 Marburg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Immekus
authors.forename: Luzi Jakob
authors.initials: LJ
authors.lastname: Barandun
authors.forename: Michael
authors.initials: M
authors.lastname: Betz
authors.forename: François
authors.initials: F
authors.lastname: Debaene
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Petiot
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanglier-Cianferani
authors.forename: Klaus
authors.initials: K
authors.lastname: Reuter
authors.forename: François
authors.initials: F
authors.lastname: Diederich
authors.forename: Gerhard
authors.initials: G
authors.lastname: Klebe
mesh_terms: D018360:Crystallography, X-Ray; D055808:Drug Discovery; D004405:Dysentery, Bacillary / Q000382:microbiology; D006801:Humans; D008958:Models, Molecular; D010430:Pentosyltransferases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects*; D012343:RNA, Transfer / Q000378:metabolism*; D012760:Shigella / Q000201:enzymology; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D017355:Zymomonas / Q000737:chemistry / Q000201:enzymology*
chemical_list: D054852:Small Molecule Libraries; D012343:RNA, Transfer; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase