Glucosylation of Ras by Clostridium sordellii lethal toxin: consequences for effector loop conformations observed by NMR spectroscopy.
The lethal toxin (LT) from Clostridium sordellii, which belongs to the family of large clostridial cytotoxins, 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 that occur upon glucosylation of the effector domain amino acid threonine-35 by LT. (31)P NMR experiments recorded during the enzymatic glucosylation process, using UDP-glucose as a cosubstrate, show that the modification of the threonine side chain influences the chemical shifts of the phosphate groups of the bound nucleotides. In the diphosphate-bound form (Ras.GDP) glucosylation of Thr35 induces only small changes in the chemical environment of the active center. In the triphosphate form with the GTP analogue GppNHp bound (Ras.GppNHp) Ras shows at least two different conformations in the active center that exchange on a medium-range time scale (10 to 0.1 ms). Glucosylation selectively stabilizes one distinct conformation 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 to effector proteins such as Raf-1, AF-6, or Byr2 and thus prevents the transduction of the activation signal in the Ras-mediated pathway. NMR correlation spectra of Ras(T35glc).GDP and denaturation experiments with urea indicate that the glucose is bound in the alpha-anomeric form to the hydroxyl group of the threonine-35 side chain. Inhibition of the glucosylation reaction by 1,5-gluconolactone suggests a stereospecific reaction mechanism with a glucosyl oxonium ion transition state for the enzymatic activity of LT.
Geyer
Matthias
M
Max-Planck-Institut für molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
Wilde
Christian
C
Selzer
Jörg
J
Aktories
Klaus
K
Kalbitzer
Hans Robert
HR
D001427:Bacterial Toxins / Q000737:chemistry*; D001665:Binding Sites; D003013:Clostridium / Q000737:chemistry*; D005942:Gluconates / Q000737:chemistry; D005947:Glucose / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000737:chemistry; D006601:Hexoses / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007783:Lactones; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010759:Phosphorus Isotopes / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D014508:Urea / Q000737:chemistry; D020663:ras Guanine Nucleotide Exchange Factors / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism*
D001427:Bacterial Toxins; D005942:Gluconates; D006601:Hexoses; D007783:Lactones; D010759:Phosphorus Isotopes; D011994:Recombinant Proteins; C072293:hemorrhagic toxin HT, Clostridium sordellii; D020663:ras Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D013912:Threonine; D006165:Guanylyl Imidodiphosphate; D014508:Urea; D018631:ras Proteins; D005947:Glucose; C010730:beta-glucono-1,5-lactone