Abstract Details 112

Production and Characterization of the Full-length LRH-1-DNA Complex
Abstract ID 112
Presenter Debanu Das
Presentation Type Poster
Full Author List

Laura Caboni1Debanu Das2,3, Fumiaki Yumoto1,4,5, Phuong Nguyen1,  Elena Sablin1, Mike Hornsby6, Jim Wells6, Marc-André Elsliger2,7, Bruce R. Conklin4, Ashley M. Deacon2,3, Ian A. Wilson2,7 and Robert J. Fletterick1


1Department of Biochemistry and Biophysics, University of California, San Francisco; 2Joint Center for Structural Genomics, http://www.jcsg.org; 3Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park; 4Gladstone Institute of Cardiovascular Disease, San Francisco; 5Structural Biology Research Center, KEK High Energy Accelerator Research Organization, Oho 1-1, Tsukuba 305-0801,Japan; 6Department of Pharmaceutical Chemistry and Cellular & Molecular Pharmacology, University of California, San Francisco; 7Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla


The transcription factor Liver Receptor Homolog-1 (LRH-1) is an important nuclear receptor in steroidogenesis, bile acid homeostasis, cholesterol transport and stem cell pluripotency. Much is known about the LRH-1 Ligand Binding Domain (LBD) and the DNA-Binding Domain (DBD), including complexes with co-activators, ligands or DNA, but there is no information on the architecture of the full-length LRH-1, which functions as a monomer. As a result, it is not known how the different domains of the full-length LRH-1 are juxtaposed to each other. In contrast, structures have been determined for some other full-length nuclear receptors, which are homodimeric or heterodimeric. The main hindrance to structural studies of full-length LRH-1 has been the availability of sufficient quantities of stable, soluble protein. We describe the production, purification and stabilization of the full-length LRH-1 in complex with the alpha inhibin promoter; functional characterization by Differential Scanning Fluorimetry (DSF) and Biolayer Interferometry (BLI); and structural characterization by Small Angle X-ray Scattering (SAXS). Additional ternary complexes with antibody fragments designed as targeted molecular tools to study LRH-1 function have been produced and similarly characterized. Our results reveal an extended molecular architecture of the functional LRH-1 that is supported by our SAXS studies on the homologous nuclear receptor, Steroidogenic Factor-1 (SF-1), which lacks an N-terminal domain, in complex with the same DNA duplex. Crystallization trials and preliminary negative stain Electron Microscopy (EM) studies for these complexes are in progress with the ultimate goal of obtaining high resolution crystal or cryoEM structures



Funding Acknowledgement