ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

Z-DNA binding protein 1 (ZBP1) belongs to a family of proteins that contain the Zα domain, which binds specifically to left-handed Z-DNA and Z-RNA. Like all vertebrate proteins in the Zα family, it contains two Zα-like domains and is highly inducible by immunostimulation. Using circular dichroism spectroscopy and electrophoretic mobility shift assays we show that both Zα domains can bind Z-DNA independently and that substrate binding is greatly enhanced when both domains are linked. Full length ZBP1 and a prominent splice variant lacking the first Zα domain (ΔZα) showed strikingly different subcellular localizations. While the full length protein showed a finely punctate cytoplasmatic distribution, ZBP1ΔZα accumulated in large cytoplasmic granules. Mutation of residues important for Z-DNA binding in the first Zα domain resulted in a distribution comparable to that of ZBP1ΔZα. The ZBP1ΔZα granules are distinct from stress granules (SGs) and processing bodies but dynamically interacted with these. Polysome stabilization led to the disassembly of ZBP1ΔZα granules, indicating that mRNA are integral components. Heat shock and arsenite exposure had opposing effects on ZBP1 isoforms: while ZBP1ΔZα granules disassembled, full length ZBP1 accumulated in SGs. Our data link ZBP1 to mRNA sorting and metabolism and indicate distinct roles for ZBP1 isoforms.     

Essential Role of the C-Terminal Helical Domain in Active Site Formation of Selenoprotein MsrA from Clostridium oremlandii

We previously determined the crystal structures of 1-Cys type selenoprotein MsrA from Clostridium oremlandii (CoMsrA). The overall structure of CoMsrA is unusual, consisting of two domains, the N-terminal catalytic domain and the C-terminal distinct helical domain which is absent from other known MsrA structures. Deletion of the helical domain almost completely abolishes the catalytic activity of CoMsrA. In this study, we determined the crystal structure of the helical domain-deleted (ΔH-domain) form of CoMsrA at a resolution of 1.76 Å. The monomer structure is composed of the central rolled mixed β-sheet surrounded by α-helices. However, there are significant conformational changes in the N- and C-termini and loop regions of the ΔH-domain protein relative to the catalytic domain structure of full-length CoMsrA. The active site structure in the ΔH-domain protein completely collapses, thereby causing loss of catalytic activity of the protein. Interestingly, dimer structures are observed in the crystal formed by N-terminus swapping between two molecules. The ΔH-domain protein primarily exists as a dimer in solution, whereas the full-length CoMsrA exists as a monomer. Collectively, this study provides insight into the structural basis of the essential role of the helical domain of CoMsrA in its catalysis.     

Shape-selective recognition of DNA abasic sites by metallohelices: inhibition of human AP endonuclease?1

Loss of a base in DNA leading to creation of an abasic (AP) site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously or under the action of various physical and chemical agents. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of AP sites in synthetic duplexes. We report here on interactions of diastereomerically pure metallo–helical ‘flexicate’ complexes, bimetallic triple-stranded ferro-helicates [Fe₂(NN-NN)₃]⁴⁺ incorporating the common NN–NN bis(bidentate) helicand, with short DNA duplexes containing AP sites in different sequence contexts. The results show that the flexicates bind to AP sites in DNA duplexes in a shape-selective manner. They preferentially bind to AP sites flanked by purines on both sides and their binding is enhanced when a pyrimidine is placed in opposite orientation to the lesion. Notably, the Λ-enantiomer binds to all tested AP sites with higher affinity than the Δ-enantiomer. In addition, the binding of the flexicates to AP sites inhibits the activity of human AP endonuclease 1, which is as a valid anticancer drug target. Hence, this finding indicates the potential of utilizing well-defined metallo–helical complexes for cancer chemotherapy.