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2002 Scientific Report

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Laboratory of Structural

Laboratory of Structural Sciences H. Eric Xu, Ph.D. Dr. Xu went to Duke University and the University of Texas Southwestern Medical Center, where he earned his Ph.D. in molecular biology and biochemistry. Following a postdoctoral fellowship with Carl Pabo at MIT, Dr. Xu moved to GlaxoWellcome in 1996, where he solved the crystal structures of a number of nuclear hormone receptors. Until recently he was a senior research investigator of nuclear receptor drug discovery at GlaxoSmithKline in Research Triangle Park, North Carolina. Dr. Xu joined VARI as a Senior Scientific Investigator in July 2002. Research Projects Our laboratory is using x-ray crystallography to study structures of key protein complexes that are important in various signaling pathways, as well as for drug discovery relevant to human diseases such as cancers and diabetes. Our major focus is on the superfamily of nuclear hormone receptors, which includes receptors for classic steroid hormones such as estrogen, progesterone, androgens, and glucocorticoids, as well as receptors for proxisome proliferator activators, vitamin D, vitamin A, and thyroid hormones. These receptors are DNA-binding and ligand-dependent transcriptional factors that regulate genes essential for a broad aspect of human physiology, ranging from development and differentiation to metabolic homeostasis. A typical nuclear receptor contains three major domains: an N-terminal activation function-1 domain (AF-1), a central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD). In addition to its role in ligand recognition, the LBD also contains dimerization motifs and an activation function-2 domain that is highly dependent on the bound ligand. The LBD is thus the key to the ligand-dependent regulation of nuclear receptor signaling and as such has been the focus of intense structural studies. One set of receptors we will study is that of the peroxisome proliferator–activated receptors, alpha, delta, and gamma, (PPARα, δ, and γ). As nuclear receptors, PPARs are regulated by the binding of small-molecule ligands. In response to ligand binding, PPARs affect a wide range of biological activities, including the regulation of lipid metabolism and of glucose homeostasis. Thus, PPARs are important therapeutic targets for common diseases such as diabetes, cancer, and cardiovascular disease. Millions of people have benefited from treatment with novel PPARγ medicines for type II diabetes; two of the top 100 drugs in 2001 were PPARγ ligands, having combined sales of over .4 billion. This demonstrates that studying PPARs can have a major effect on human health and disease treatment. To understand the molecular basis of ligand-mediated signaling by PPARs, we have determined crystal structures of each PPAR’s LBD (Figure 1) Figure 1. Crystal structure of the PPAR LBDs and their ligand-binding pocket. Each PPAR contains 13 α-helices and 4 small β-strands. The helices are arranged into a three-layer sandwich fold to create a ligand-binding pocket (white surface). The ability of PPARs to recognize so many and such diverse ligands can be accounted by the enormous size of the PPAR pocket, which is over 1,300 Å 3 and is much larger than the pocket seen in any other nuclear receptor. bound to many diverse ligands, including fatty acids, the lipid-lowering drugs called fibrates, and a new generation of antidiabetic drugs, glitazones. These structures have provided a framework for understanding the mechanisms of ago- 59

Figure 2. Crystal structure of the human GR LBD bound to dexamethasone and a TIF2 co-activator. This structure reveals a novel dimer configuration, a second charge clamp, and a unique GR pocket. The GR LBD dimer interface, composed of β-loops and turns, is distinct from the helix-10 interface of the RXR/PPARγ heterodimer or the RXR homodimer. nists and antagonists, as well as the recruitment of co-activators and co-repressors in gene activation and repression. Furthermore, these structures also serve as a molecular basis for understanding potency, selectivity, and binding mode of diverse ligands, information that has provided critical insights for designing the next generation of PPAR medicines. The other nuclear receptor we will study is the human glucocorticoid receptor (GR). GR plays key roles in the metabolism of lipids and carbohydrates, development of the central nervous system, and homeostasis of the immune system. GR is also associated with numerous pathological pathways and as such is an important drug target. In fact, GR has a rich history in drug discovery and human medicine. There are more than 10 GR ligands (including dexamethasone) that are currently used for treatment of such diverse medical conditions as asthma, allergies, autoimmune diseases, and cancer. At the molecular level, GR can function either as a transcription activator or a transcription repressor. Both of these functions of GR are tightly regulated by small ligands that bind to the GR ligand-binding domain. To explore the molecular mechanism of ligand binding and signaling of GR, we have determined a crystal structure of the GR LBD bound to dexamethasone and a co-activator motif from TIF2. The structure reveals a novel LBD–LBD dimer interface (Figure 2) that is crucial for GR-mediated transactivation but not transrepression, suggesting a novel role of LBD dimerization in the GR signaling pathways. The structure also contains an unexpected charge clamp responsible for sequence-specific binding of co-activators and a unique ligand-binding pocket to account for specific recognition of diverse GR ligands. The detailed molecular interactions between the receptor and dexamethasone should also facilitate the discovery of new glucocorticoid receptor molecules that would reduce the side effects of current GR drugs. Beside PPARs and GR, the human genome contains 44 other nuclear receptors. X-ray structures have now been solved for more than a dozen LBDs, bound to agonists and antagonists, co-activators and co-repressors, and in forms of monomers, homodimers, heterodimers, and tetramers. These structures have illustrated the details of ligand binding, the conformational changes induced by agonists and antagonists, the basis of dimerization, and the mechanism of coactivator and co-repressor binding. All of the nuclear receptors studied to date have broadly similar structures and mechanisms of activation, but functionally significant differences have arisen among the various receptors over the course of evolution. We can expect more surprises as structural work continues on the remaining nuclear receptors, and as crystallographers tackle higherorder complexes involving the LBD with the AF- 1 domain, the DBD, and other proteins and nucleic acids involved in gene transcription. 60

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