The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A-HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a "lock and key" interaction is typical of innate receptor-ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.
Recently, there have been numerous reports on the use of different fluorescent DNA stains for specific minor groove binding. The exploration of biological markers increases the safety of their use as diagnostic criteria. Single crystal analysis of DNA–ligand binding interactions is of essential importance to obtain the requirements for their usage in the pharmaceutical and medical industries. Dyes that bind to DNA, such as Hoechst 33342 or 4′,6-diamidino-2-phenylindole (DAPI), can be used not only for analytical use, but for medical purposes. DAPI and Hoechst 33342 are fluorescent dyes that bind to the minor groove of DNA, fluorescing brightly in the blue region with an emission maximum at approximately 461 nm when excited by ultraviolet light (~350 nm). This work focuses on the binding interactions of Hoechst 33342 with the specific DNA sequence d(CGTGAATTCACG)2. The structure of the complex was determined using single-crystal X-ray diffraction at a resolution of 1.9 Å in the space group P212121. The coordinates and structure factors are deposited in the RCSB Protein Data Bank (PDB) under entry 9FT8. The structure is nearly isomorphous with that of previously reported crystal structures of the oligonucleotide d(CGTGAATTCACG)2 alone (PDB ID: 5JU4) and with that in complexes with DAPI (5T4W). The adjustments in crystal interactions between the native DNA molecule and the DNA–DAPI complex are described. Hoechst 33342 selectively binded to the tight minor groove close to the midpoint of the B-DNA segment, adjacent to the A–T base pairs. It interacted with DNA through hydrogen bonding and van der Waals forces. The structural comparison revealed that Hoechst 33342 inserts itself in the minor groove in a strongly specific manner, displacing the ordered spine waters.
