My research interests are concerned with the three-dimensional structures of proteins and the relevance of these structures in biology. The laboratory utilizes a combination of biophysics, biochemistry and techniques from cell biology. We use a broad range of tools that include X-ray crystallography for protein structure determination, molecular biology (heterologous over-expression, & site-directed mutagenesis), protein engineering and biochemistry. We combine the static data provided by protein crystallography with techniques of cell biology that can monitor the activity of proteins, fluorescence and nuclear magnetic resonance spectroscopy that can monitor the conformational dynamics of these molecules and isothermal titration calorimetry that provides a direct measure of the interactions between molecules and associated thermodynamic parameters. We also collaborate with Prof. Pat Griffin (Scripps Institute) to measure rates of hydrogen-deuterium exchange by mass spectrometry. These methods are applied to several biological problems that are initiated by binding of hormones to cellular receptors and the transduction of these signals.
Within this laboratory we explore the basis of allosterism within nuclear hormone receptors. Hormonal molecules such as glucocorticoids, retinoids, thyroid and vitamin-derived hormones exert their effects by regulating the transcription of hormone-responsive target genes within the nucleus of cells. These hormones function by directly binding to and modulating the activity of this nuclear receptor family of transcription factors. These hormone-regulated proteins direct virtually every aspect of human physiology and improper function can lead to several disease states such as prostate and breast cancer, diabetes, obesity, heart disease, osteoporosis, and processes associated with aging. Despite the wealth of data on the genetics and cellular localization of nuclear receptors, there is relatively little known of the precise molecular mechanisms of regulation of these proteins. There is an emerging view that nuclear receptor proteins populate a wide ensemble of fluctuating conformations. These conformational movements result from intrinsic structural motifs and interactions with regulatory molecules. Such motifs and interacting molecules can provide additional layers of regulation by being allosterically coupled to functional sites through distinct structural pathways. These mechanisms may explain the complexity of the behavior of nuclear receptors in various tissue types or on different DNA elements. My lab is developing models for nuclear receptor allosterism that address the following questions: (i) By what mechanisms do discrete sites regulate the activity of these modular receptors, as noticed in some disease-causing mutants? (ii) How do the DNA and ligand binding domains (DBD and LBD) communicate to allosterically regulate coactivator and DNA recognition? (iii) What allosteric role do cofactors that bind the amino terminus play in receptor regulation? (iv) What is the molecular basis at the level of residue interactions for these allosteric processes?
Ours was the first laboratory to determine the three-dimensional structure of murine constitutive androstane receptor (CAR) ligand binding domain. This is the first novel protein structure from East Tennessee. Additionally, we have recently determined the structure of the ligand binding domain of the thyroid receptor:retinoid X receptor (TR:RXR) complex, also by X-ray crystallography.
Putcha, B.-D. K., Wright, E., Brunzelle, JS, and Elias J. Fernandez*. Structural basis for negative cooperativity within agonist-bound TR:RXR heterodimers. (2012) Proc. Natl. Acad. Sci. (USA). 1119852109.pdf
Edward Wright, Scott A. Busby, Sarah Wisecarver, Jeremy Vincent, Patrick R. Griffin, Elias J. Fernandez*. (2011) Helix 11 Dynamics Is Critical for Constitutive Androstane Receptor Activity, Structure (Cell Press) Jan 12;19(1):37-44.
Putcha, B.-D. K. and Elias Fernandez*. Direct inter-domain interactions can mediate allosterism in the thyroid receptor.(2009) J. Biol. Chem.(ASBMB) 21;284(34):22517-24.
Wright Edward, Vincent J, Fernandez EJ*. Thermodynamic Characterization of the Interaction Between CAR/RXR and SRC-1 Peptide by Isothermal Titration Calorimetry.(2007) Biochemistry (ACS) 46(3):862-70.
Vincent J, Shan L, Fan M, Brunzelle JS, Forman BM and Fernandez E (2005) Crystallographic analysis of murine constitutive androstane receptor ligand-binding domain complexed with 5alpha-androst-16-en-3alpha-ol. Acta Cryst. (IUCr) F61:156-159.
Shan L, Vincent J, Brunzelle JS, Dussault I, Lin M, Ianculescu I, Sherman MA, Forman BM and Fernandez EJ (2004) Structure of the murine constitutive androstane receptor complexed to androstenol; a molecular basis for inverse agonism. Molecular Cell (Cell Press) Dec 22; 16(6):907-17.