NMR structural studies of the human protein DEK

The chromatin-associated human DEK protein was first identified as a fusion protein in patients with acute myeloid leukemia and has since been implicated in many diseases, but as yet no clear biochemical function has been elucidated. A structural biology approach was taken to characterize the biochemical properties of DEK with the goal of providing insight into its cellular role(s).; Solution NMR was used to characterize the structural, dynamics and DNA binding properties of human DEK. Through an iterative process of production and study of fragments of DEK, starting from an inspection of the primary sequence of the protein, it was discovered that DEK contains two domains with well-defined secondary structure, an N-terminal domain (DEK78--187) and a C-terminal domain (DEK309--375). The three-dimensional structures for these two domains were determined using triple resonance experiments, and HSQC chemical shift mapping was used to explore their DNA binding characteristics.; In the entire 375 amino-acids of the DEK protein, only one short sequence (35 residues) located in the N-terminal domain contains sequence homology to other known proteins: the putative DNA-binding motif, SAP. This domain (DEK78--187) consists of 5 alpha-helices arranged as two helix-extended loop-helix (HEH) motifs facing each other, one HEH being the SAP motif, the other was found to contain a second DNA binding site.; The C-terminal domain is known to be the antigenic fragment in juvenile rheumatoid arthritis and can complement the phenotype of cells derived from ataxia-telangiectasia patients. This domain (DEK309--375) was determined to contain a 3 alpha-helix bundle with a right-handed twist. This fold is reminiscent of the winged helix fold of DNA binding proteins. DNA-binding studies indicated that this domain binds to double-stranded DNA using the second helix of the bundle.; Like the primary sequence, the tertiary structures of the domains of DEK are themselves relatively unique in biology. Database searches discovered only protein structures with slight similarity---all DNA-binding protein domains. Additionally, the DNA-binding studies indicated that the N-terminal domain, DEK78--187, has a novel DNA-binding mechanism responsible for the DNA supercoiling activity of wild-type DEK.