The Importance of Charge in Perturbing the Aromatic Glue Stabilizing the Protein-Protein Interface of Homodimeric tRNA-Guanine Transglycosylase.

Abstract

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine, and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since it is only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each mutated variant with respect to the dimer and thermal stability or enzyme activity by applying native mass spectrometry, a thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate a strong influence of pH on the homodimer stability. Apparently, protonation of a histidine within the aromatic cluster supports the collapse of an essential structural motif within the dimer interface at slightly acidic pH.

• Authors: Andreas Nguyen, Dzung Nguyen, Tran Xuan Phong Nguyen, Maurice Sebastiani, Stefanie Dörr, Oscar Hernandez-Alba, François Debaene, Sarah Cianférani, Andreas Heine, Gerhard Klebe, Klaus Reuter
• Journal: ACS chemical biology (ACS Chem Biol) Vol. 15 Issue 11 Pg. 3021-3029 (11 20 2020) ISSN: 1554-8937 [Electronic] United States
• PMID: 33166460 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Pentosyltransferases
• queuine tRNA-ribosyltransferase

Topics

• Catalytic Domain
• Crystallography, X-Ray
• Enzyme Stability
• Models, Molecular
• Mutation
• Pentosyltransferases (chemistry, genetics)
• Protein Conformation
• Protein Multimerization
• Zymomonas (chemistry, enzymology, genetics)