Glucosylation of Ras by Clostridium sordellii Lethal Toxin: Consequences for Effector Loop Conformations Observed by NMR Spectroscopy
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- 作者：Matthias Geyer ; Christian Wilde ; Jö ; rg Selzer ; Klaus Aktories ; and Hans Robert Kalbitzer
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：October 21, 2003
- 年：2003
- 卷：42
- 期：41
- 页码：11951 - 11959
- 全文大小：283K
- 年卷期：v.42,no.41(October 21, 2003)
- ISSN：1520-4995
文摘
The lethal toxin (LT) from Clostridium sordellii, which belongs to the family of large clostridialcytotoxins, acts as a monoglucosyltransferase for the Rho subfamily GTPase Rac and also modifies Ras.In the present study we investigated structural changes of H-Ras in its di- and triphosphate form thatoccur upon glucosylation of the effector domain amino acid threonine-35 by LT. 31P NMR experimentsrecorded during the enzymatic glucosylation process, using UDP-glucose as a cosubstrate, show that themodification of the threonine side chain influences the chemical shifts of the phosphate groups of thebound nucleotides. In the diphosphate-bound form (Ras·GDP) glucosylation of Thr35 induces only smallchanges in the chemical environment of the active center. In the triphosphate form with the GTP analogueGppNHp bound (Ras·GppNHp) Ras shows at least two different conformations in the active center thatexchange on a medium-range time scale (10 to 0.1 ms). Glucosylation selectively stabilizes one distinctconformation of the effector loop (state 1) with tyrosine-32 probably apart from the nucleotide and threonine-35 not involved in magnesium ion coordination. This conformation is known to have a low affinity toeffector proteins such as Raf-1, AF-6, or Byr2 and thus prevents the transduction of the activation signalin the Ras-mediated pathway. NMR correlation spectra of Ras(T35glc)·GDP and denaturation experimentswith urea indicate that the glucose is bound in the 
-anomeric form to the hydroxyl group of the threonine-35 side chain. Inhibition of the glucosylation reaction by 1,5-gluconolactone suggests a stereospecificreaction mechanism with a glucosyl oxonium ion transition state for the enzymatic activity of LT.
-anomeric form to the hydroxyl group of the threonine-35 side chain. Inhibition of the glucosylation reaction by 1,5-gluconolactone suggests a stereospecificreaction mechanism with a glucosyl oxonium ion transition state for the enzymatic activity of LT.