DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (K(D) ∼60 μM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.

DAXX is a scaffold protein with diverse roles that often depend upon binding SUMO via its N-and/or C-terminal SUMO-interacting motifs (SIM-N and SIM-C). Using NMR spectroscopy, we characterized the in vitro binding properties of peptide models of SIM-N and SIM-C to SUMO-1 and SUMO-2. In each case, binding was mediated by hydrophobic and electrostatic interactions and weakened with increasing ionic strength. Neither isolated SIM showed any significant paralog specificity, and the measured M range K D values of SIM-N toward both SUMO-1 and SUMO-2 were ϳ4-fold lower than those of SIM-C. Furthermore, SIM-N bound SUMO-1 predominantly in a parallel orientation, whereas SIM-C interconverted between parallel and antiparallel binding modes on an ms to s time scale. The differences in affinities and binding modes are attributed to the differences in charged residues that flank the otherwise identical hydrophobic core sequences of the two SIMs. In addition, within its native context, SIM-N bound intramolecularly to the adjacent N-terminal helical bundle domain of DAXX, thus reducing its apparent affinity for SUMO. This behavior suggests a possible autoregulatory mechanism for DAXX. The interaction of a C-terminal fragment of DAXX with an N-terminal fragment of the sumoylated Ets1 transcription factor was mediated by SIM-C. Importantly, this interaction did not involve any direct contacts between DAXX and Ets1, but rather was derived from the non-covalent binding of SIM-C to SUMO-1, which in turn was covalently linked to the unstructured N-terminal segment of Ets1. These results provide insights into the binding mechanisms and hence biological roles of the DAXX SUMOinteracting motifs.DAXX is an enigmatic protein with diverse and often controversial roles. Although first discovered through a screen for association with the cytoplasmic Fas death domain (1), DAXX is now recognized to function primarily in the nucleus and to often be localized within subnuclear structures known as promyelocytic leukemia nuclear bodies (PML-NBs) 3 (2, 3). Perhaps the best established role of DAXX lies with the regulation of transcription factors. Although some of these factors may be activated by DAXX (4, 5), in most cases, DAXX is linked with transcriptional repression (3). DAXX also associates with histone deacetylases as well as other chromatin-remodeling systems, thereby providing plausible routes to the repression of gene expression (6 -10). The key to the localization and function of DAXX is its ability to bind the ubiquitin-like protein SUMO (3, 11).The human genome encodes four SUMO paralogs (for reviews, see Refs. 12-16). SUMO-2 and SUMO-3 are virtually identical and are generally referred to as SUMO-2/3. SUMO-1 and SUMO-2/3 have ϳ50% sequence identity and are expressed in all cells, whereas the function of SUMO-4 remains unclear. Using a common enzymatic pathway, many substrates can be covalently modified by both SUMO-1 and SUMO-2/3. However, the latter can also form chains on substrate proteins, and the levels of SUMO-2/3 conjugation ...

Current theories suggest that mitotic checkpoint proteins are essential for proper cellular response to taxanes, a widely-used family of chemotherapeutic compounds. We recently demonstrated that absence or depletion of protein Daxx increases cellular taxol (paclitaxel) resistance—a common trait of patients diagnosed with several malignancies, including breast cancer. Further investigation of Daxx-mediated taxol response revealed that Daxx is important for the proper timing of mitosis progression and cyclin B stability. Daxx interacts with mitotic checkpoint protein Rassf1 and partially co-localizes with this protein during mitosis. Rassf1/Daxx depletion or expression of Daxx binding domain of Rassf1 elevates cyclin B stability and increases taxol resistance in cells and mouse xenograft models. In breast cancer patients, we observed the inverse correlation between Daxx and clinical response to taxane-based chemotherapy. These data suggest that Daxx and Rassf1 define a mitotic stress checkpoint that enables cells to exit mitosis as micronucleated cells (and eventually die) when encountered with specific mitotic stress stimuli, including taxol. Surprisingly, depletion of Daxx or Rassf1 does not change activity of E3 ubiquitin ligase APC/C in in vitro settings, suggesting necessity of mitotic cellular environment for proper activation of this checkpoint. Daxx and Rassf1 may become useful predictive markers for the proper selection of patients for taxane chemotherapy.

In Escherichia coli, the BAM complex catalyzes the essential process of assembling outer membrane proteins (OMPs). This complex consists of five proteins: one membrane-bound protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their importance in OMP biogenesis, there is currently a lack of functional and structural information on the BAM complex lipoproteins. BamE is the smallest but most conserved lipoprotein in the complex. The structural and dynamic properties of monomeric BamE (residues 21-133) were determined by NMR spectroscopy. The protein folds as two α-helices packed against a three-stranded antiparallel β-sheet. The N-terminal (Ser21-Thr39) and C-terminal (Pro108-Asn113) residues, as well as a β-hairpin loop (Val76-Gln89), are highly flexible on the subnanosecond time scale. BamE expressed and purified from E. coli also exists in a kinetically trapped dimeric state that has dramatically different NMR spectra, and hence structural features, relative to its monomeric form. The functional significance of the BamE dimer remains to be established. Structural comparison to proteins with a similar architecture suggests that BamE may play a role in mediating the association of the BAM complex or with the BAM complex substrates.