An integrative approach combining noncovalent mass spectrometry, enzyme kinetics and X-ray crystallography to decipher Tgt protein-protein and protein-RNA interaction.
The tRNA-modifying enzyme tRNA-guanine transglycosylase (Tgt) is a putative target for new selective antibiotics against Shigella bacteria. The formation of a Tgt homodimer was suggested on the basis of several crystal structures of Tgt in complex with RNA. In the present study, noncovalent mass spectrometry was used (i) to confirm the dimeric oligomerization state of Tgt in solution and (ii) to evidence the binding stoichiometry of the complex formed between Tgt and its full-length substrate tRNA. To further investigate the importance of Tgt protein-protein interaction, point mutations were introduced into the dimer interface in order to study their influence on the formation of the catalytically active complex. Enzyme kinetics revealed a reduced catalytic activity of these mutated variants, which could be related to a destabilization of the dimer formation as evidenced by both noncovalent mass spectrometry and X-ray crystallography. Finally, the effect of inhibitor binding was investigated by noncovalent mass spectrometry, thus providing the binding stoichiometries of Tgt:inhibitor complexes and showing competitive interactions in the presence of tRNA. Inhibitors that display an influence on the formation of the dimer interface in the crystal structure are promising candidates to alter the protein-protein interaction, which could provide a new way to inhibit Tgt.
Ritschel
Tina
T
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35032 Marburg, Germany.
Atmanene
Cédric
C
Reuter
Klaus
K
Van Dorsselaer
Alain
A
Sanglier-Cianferani
Sarah
S
Klebe
Gerhard
G
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray / Q000379:methods; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D007535:Isomerases / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D055503:Protein Multimerization; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D006147:Guanine; C019549:queuine; D012343:RNA, Transfer; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase; C082572:S-adenosylmethionine - tRNA ribosyltransferase-isomerase; D007535:Isomerases