UC San Diego

Two proteins important in transcriptional regulation and apoptotic modulation are RelB and Daxx. Previously, it was shown that Daxx induced apoptosis through interaction with RelB to silence Daxx transcriptional activity. The main focus of this study was to identify the binding domains required for such interaction and to purify the recombinant RelB:Daxx heterodimer from E.coli for x-ray crystallography analysis. The findings showed that Daxx DHBI (56-144) region interacts with RelB DD (277-391). This finding was confirmed through GST pulldown analysis with different fragments of Daxx and RelB. Some of the RelB DD mutants (I335F; I286M, I335F; and Y300F, I335F) which can enhance the binding ability of RelB DD were shown to abrupt the interaction with Daxx DHBI. Surprisingly, it was found that the strength of interaction with RelB RHR (1-400) and Daxx DHBI was not strong enough to be able to be purified as a single peak complex via gel filtration. While Daxx and RelB are known to interact with each other to induce apoptosis, the binding affinity is not strong enough perhaps due to competition with other NF[kappa]B factors binding to the open interface of RelB DD. In order to get a more stable Daxx:RelB complex, future studies can focus on the expression and purification of Daxx N-terminus (1-400) and RelB RHR (1-400) complex. Also, purification and crystallization of RelB DD mutants can give better understanding about the dimerization domain behavior and as well as their effect in binding to Daxx