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Additional file 3. Peer review history.

Additional file 1: Figure S1-S4. Supplementary figure legends and supplementary figures.

Partial funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund., DNA can form many structures beyond the canonical Watson–Crick double helix. It is now clear that noncanonical structures are present in genomic DNA and have biological functions. G-rich G-quadruplexes and C-rich i-motifs are the most well-characterized noncanonical DNA motifs that have been detected in vivo with either proscribed or postulated biological roles. Because of their independent sequence requirements, these structures have largely been considered distinct types of quadruplexes. Here, we describe the crystal structure of the DNA oligonucleotide, d(CCAGGCTGCAA), that self-associates to form a quadruplex structure containing two central antiparallel G-tetrads and six i-motif C–C+ base pairs. Solution studies suggest a robust structural motif capable of assembling as a tetramer of individual strands or as a dimer when composed of tandem repeats. This hybrid structure highlights the growing structural diversity of DNA and suggests that biological systems may harbor many functionally important non-duplex structures.

International audience; Ubiquitination is a critical protein post-translational modification involved in a variety of vital processes in eukaryotic cells. The discovery of ubistatins [1], small molecules that impair proteasomal degradation of proteins by directly binding to (poly)ubiquitin upstream of the proteasome, makes ubiquitin itself a potential therapeutic target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural details of ubiquitin-ubistatin interactions has impeded their development. To address this deficiency, a panel of new ubistatin derivatives was synthesized and characterized using functional and NMR-based binding assays [2]. We found that the most active compounds contain strongly acidic groups. We then used NMR and small-angle neutron scattering (SANS) to determine the structures of ubiquitin complexes with ubistatin B and hemi-ubistatin B. These structures revealed direct interactions of ubistatins with ubiquitin’s hydrophobic surface-patch and the basic/polar residues surrounding it, which were confirmed by site-directed mutagenesis. Our results show that ubistatin B binds ubiquitin and di-ubiquitin tighter than a high-affinity ubiquitin-receptor, the UBA domain from the proteasomal shuttle protein ubiquilin-1, and shows clear preference for ubiquitin chains linked via K48 over those linked via K11 or K63. The 15N relaxation and SANS data revealed unexpected binding stoichiometries and structural arrangements of ubiquitin or di-ubiquitins in those complexes. Furthermore, through binding to ubiquitin, ubistatin B shields ubiquitin conjugates from disassembly by a range of deubiquitinases, including the 26S proteasome.Finally, we found that ubistatin B penetrates human cancer cells and perturbs the cellular ubiquitin landscape. These findings highlight versatile properties of ubistatins and have implications for their future development and use in targeting ubiquitin-mediated signaling pathways. Combined with the earlier observations that ubistatins can arrest the cell cycle, producing effects similar to proteasome inhibitors [1]), our structural data suggest that the ubiquitin signal is a plausible candidate for therapeutic intervention in the ubiquitin-proteasome pathway.