Furthermore, also for SUMO 1, only despite some N terminal resonances are observable while the major part of SUMO 1 resonances are too broad to be detected, somewhat mimicking the NMR behavior of TDG CAT Inhibitors,Modulators,Libraries and TDG RD domains. These data are con sistent with the X ray structure of TDG conjugated to SUMO1 where tight associations between SUMO 1 and TDG CAT through the C terminal SBM were high lighted. The resonances of the TDG N terminal TDG with DNA as well as sumoylation Inhibitors,Modulators,Libraries of TDG prevent further SUMO 1 intermolecular interactions. The non covalent interactions with SUMO 1 could be either implicated in the TDG sumoylation process itself as intermediate states, or in functional interactions between TDG and other sumoylated proteins.

Moreover, since SUMO conjugation to TDG was shown to reduce its DNA binding activity, which suggests when seen in context of previous works, a putative modification of the TDG N terminal conformation, we have investigated the intermolecular inter actions between TDG and SUMO 1 by NMR spectro scopy. Brefeldin_A In direct binding experiments, we have not detected chemical shift perturbations of the resonances of the isolated N terminal domain in the presence of a 3 fold excess of SUMO 1. These data confirm that there are no direct interactions between SUMO 1 and the N terminal domain of TDG. Moreover, in 15N labeled full length TDG, the resonances Inhibitors,Modulators,Libraries of the regulatory domain become partially detectable upon unlabeled SUMO 1 addition while no modification was detected for the first fifty N terminal residues.

We indeed show a number of new resonances on the 15 N 1H HSQC spectrum of the 15N labeled TDG pro region are not perturbed upon SUMO 1 conjugation Inhibitors,Modulators,Libraries when compared to non modified TDG pro tein. In contrast, the resonances of residues 327 to 347, surrounding the K330 sumoylation site, are significantly broadened, indicating conformational modifica tions of the TDG C terminus through covalent sumoyla tion and no remote perturbations of the N terminal conformation. We cannot exclude, given the absence of detectable NMR signals that some conformational changes of the TDG regulatory and catalytic domains upon SUMO 1 conjugation occur. Note, however, that based on previous work a structural change of at least the TDG active site after SUMO conjugation is rather unlikely.

TDG SUMO 1 non covalent interactions induce conformational changes within the N terminal regulatory domain and the C terminal region of TDG It had previously been shown that SUMO 1 can interact with TDG also in a non covalent manner through apparently two distinct binding sites Vismodegib price located within TDG CAT and that the interactions of tein in the presence of SUMO 1 that match very well with those of TGD RD observed in the context of the isolated TDG N terminus indicating that SUMO 1 produces a conforma tional change of TDG RD upon binding to SBMs.