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

• Published in: PloS one Vol. 12; no. 4; p. e0175723
• Main Authors: Ehrmann, Frederik Rainer, Stojko, Johann, Metz, Alexander, Debaene, François, Barandun, Luzi Jakob, Heine, Andreas, Diederich, François, Cianférani, Sarah, Reuter, Klaus, Klebe, Gerhard
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18-04-2017; Public Library of Science (PLoS)
• Subjects: Adaptations; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding sites; Biology and life sciences; Catalytic Domain; Chemotherapy; Cocrystallization; Complex formation; Crystal structure; Crystallization; Crystallography, X-Ray; Dimerization; Dimers; Dysentery; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Genetic aspects; Guanine; Hydrophobic and Hydrophilic Interactions; Inhibition; Ligands; Models, Molecular; Molecular structure; Packing; Pathogenesis; Pentosyltransferases - antagonists & inhibitors; Pentosyltransferases - chemistry; Pentosyltransferases - metabolism; Perturbation; Phosphates; Physical sciences; Physiological aspects; Post-transcription; Protein Binding; Protein Conformation; Protein Domains; Protein Multimerization; Protein Stability; Protein Structure, Secondary; Proteins; Research and Analysis Methods; Ribose; RNA, Transfer - chemistry; RNA, Transfer - genetics; RNA, Transfer - metabolism; Shigella; Shigellosis; Soaking; Solutions; Thermodynamics; Titration; Transcription; Transfer RNA; tRNA; tRNA-binding protein; tRNA-guanine transglycosylase; Virulence; Virulence (Microbiology); Water chemistry; Waterborne diseases; Zymomonas - enzymology; Switzerland; Germany; France
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0175723

For the efficient pathogenesis of Shigella, the causative agent of bacillary dysentery, full functionality of tRNA-guanine transglycosylase (TGT) is mandatory. TGT performs post-transcriptional modifications of tRNAs in the anticodon loop taking impact on virulence development. This suggests TGT as a putative target for selective anti-shigellosis drug therapy. Since bacterial TGT is only functional as homodimer, its activity can be inhibited either by blocking its active site or by preventing dimerization. Recently, we discovered that in some crystal structures obtained by soaking the full conformational adaptation most likely induced in solution upon ligand binding is not displayed. Thus, soaked structures may be misleading and suggest irrelevant binding modes. Accordingly, we re-investigated these complexes by co-crystallization. The obtained structures revealed large conformational rearrangements not visible in the soaked complexes. They result from spatial perturbations in the ribose-34/phosphate-35 recognition pocket and, consequently, an extended loop-helix motif required to prevent access of water molecules into the dimer interface loses its geometric integrity. Thermodynamic profiles of ligand binding in solution indicate favorable entropic contributions to complex formation when large conformational adaptations in the dimer interface are involved. Native MS titration experiments reveal the extent to which the homodimer is destabilized in the presence of each inhibitor. Unexpectedly, one ligand causes a complete rearrangement of subunit packing within the homodimer, never observed in any other TGT crystal structure before. Likely, this novel twisted dimer is catalytically inactive and, therefore, suggests that stabilizing this non-productive subunit arrangement may be used as a further strategy for TGT inhibition.