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Structural and functional characterization of the DAXX N-terminal helix bundle and SUMO interaction motifs Escobar-Cabrera, Eric

Abstract

The Fas death-domain associated (DAXX) protein was first discovered as an intermediary of a FADD-independent apoptosis signaling pathway. However, subsequent studies have established it as an important player in both transcription and cell cycle regulation. In this thesis, the first structural characterization of DAXX is presented. Sequence alignment and secondary structure prediction algorithms were used to define a number of constructs of DAXX. The C-terminal ~l/3 of DAXX was found to be intrinsically disordered, whereas a well-defined folded domain was identified near its N-terminus. NMR spectroscopy was used to solve the three-dimensional structure of this domain, and to characterize its dynamic behavior. The calculated structural ensemble consists of five helices, and hence is named the DAXX Helix Bundle (DHB) domain. This domain has a very different topology to the Sin3 PAH domains, which until now have been used as a model for this region of DAXX. Rassfl C, an important tumor suppressor, was reported recently to interact with DAXX via the DHB domain. This interaction was linked to mitosis progression, and has potential implications in the treatment of cancer. The NMR-derived ensemble of the complex of the DHB domain with an N-terminal fragment of Rassf1 C revealed a short amphipathic a-helix filling the cleft between helices 2 and 5 of the DHB domain. Both hydrophobic and electrostatic interactions mediated complex formation. This structural characterization explains the observed in vivo interaction and provides clues as to how the binding might be regulated. Additionally, two SUMO-interacting motifs at the termini of DAXX, SIM-N and SIM-C, were characterized. Their interactions with SUMO- 1 and SUMO-2 were examined structurally and thermodynamically using NMR spectroscopy. SIM-N was also found to bind intramolecularly to the DHB domain and SIM-C to mediate the interaction of DAXX with the sumoylated Ets- 1 transcription factor. Importantly, the latter did not involve any direct contacts between DAXX and Ets- 1, but rather derived from the non-covalent binding of DAXX SIM-C to SUMO- 1, which in turn was covalently linked in a “beads-on-a-string” fashion to Ets- 1. These results provide new insights into the binding mechanisms and biological roles of DAXX-SUMO interactions.

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