Solution structure of the Zbeta domain of human DNA-dependent activator of IFN-regulatory factors and its binding modes to B- and Z-DNAs.

The DNA-dependent activator of IFN-regulatory factors (DAI), also known as DLM-1/ZBP1, initiates an innate immune response by binding to foreign DNAs in the cytosol. For full activation of the immune response, three DNA binding domains at the N terminus are required: two Z-DNA binding domains (ZBDs), Zα and Zß, and an adjacent putative B-DNA binding domain. The crystal structure of the Zß domain of human DAI (hZß(DAI)) in complex with Z-DNA revealed structural features distinct from other known Z-DNA binding proteins, and it was classified as a group II ZBD. To gain structural insights into the DNA binding mechanism of hZß(DAI), the solution structure of the free hZß(DAI) was solved, and its bindings to B- and Z-DNAs were analyzed by NMR spectroscopy. Compared to the Z-DNA-bound structure, the conformation of free hZß(DAI) has notable alterations in the α3 recognition helix, the "wing," and Y145, which are critical in Z-DNA recognition. Unlike some other Zα domains, hZß(DAI) appears to have conformational flexibility, and structural adaptation is required for Z-DNA binding. Chemical-shift perturbation experiments revealed that hZß(DAI) also binds weakly to B-DNA via a different binding mode. The C-terminal domain of DAI is reported to undergo a conformational change on B-DNA binding; thus, it is possible that these changes are correlated. During the innate immune response, hZß(DAI) is likely to play an active role in binding to DNAs in both B and Z conformations in the recognition of foreign DNAs.

Structure of the Cdt1 C-terminal domain: conservation of the winged helix fold in replication licensing factors.

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. The C-terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2-7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X-ray crystallography and solution NMR spectroscopy, respectively. While the N-terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172-368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.