Structural Approaches To Understanding The Regulation Of

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications & trials

Abstract

The goal of our research program is to understand the mechanism of regulation of biological activity in macromolecular complexes through structural analysis, using X-ray crystallography as our primary tool. (1) Adeno-associated virus (AAV) Rep. Only one animal virus, AAV, integrates its genome into a particular location in human chromosomal DNA. This unique property has important implications for the targeted delivery of genes in the context of gene therapy vectors. Viral integration requires the AAV Rep protein, a multifunctional enzyme that possesses a site-specific DNA binding activity, an endonuclease activity, an ATPase activity, and a 3'-to-5' helicase activity. To shed light onto the mechanism of site-specific integration, we have determined the structure of the endonuclease domain of AAV5 Rep bound to the series of direct repeats that comprise the Rep binding site at the viral termini and at AAVS2, the site of integration in chromosome 19. We find five Rep monomers, each bound to a tetranucleotide repeat, spiralling along the DNA axis. DNA recognition involves a complex combination of binding interactions within both the major and minor grooves, and a given repeat is contacted by two sequential monomers with the spiral. We have also determined the structure of the endonuclease domain bound to the stem-loop sequence that occurs on one arm of the three-way DNA junction that forms the viral inverted terminal repeat. This sequence is an important element in distinguishing replicated viruses from those that remain unreplicated. (2) DNA transposition by Tn7. The Tn7 transposon is a mobile genetic element that encodes several protein molecules necessary for its excision from a carrier and its subsequent integration into the E. coli chromosome. Four Tn7 encoded proteins (TnsA,B,C, and D) work together in a highly regulated manner to ensure accurate insertion of the transposon into a particular site downstream of the E. coli glutamine synthetase gene. Our aim is to understand the structural basis of transpososome assembly and regulation. To this end, we have determined the structure of TnsA, the transposase component responsible for 5' end cleavage, bound to the C-terminal region of TnsC, the central regulatory molecule in Tn7. The structure demonstrates that the role of this region of TnsC is primarily to stabilize TnsA and to aid in the binding of TnsA to DNA. We have confirmed the former using differential scanning calorimetry, and the latter by gel retardation assays. (3) The structural biology of complexes of 14-3-3. The 14-3-3 proteins, a family of dimeric regulatory proteins, are found within all eukaryotic cells and are involved in many biological processes. During the past year, we have continued our efforts to characterize the complex between 14-3-3 and FOXO4, a transcription factor that functions in the insulin signaling pathway. We have quantitatively characterized the in vitro interactions between 14-3-3, FOXO4, and its target DNA, the insulin response element. These studies have demonstrated that doubly phosphorylated FOXO4 binds 14-3-3 with a Kd of less than 30 nM, and that formation of this complex results in the complete inhibition of FOXO4 binding to DNA. Crystallographic studies continue.