Randy K. Bledsoe

Crystal Structure of the Glucocorticoid Receptor Ligand Binding Domain Reveals a Novel Mode of Receptor Dimerization and Coactivator Recognition

Transcriptional regulation by the glucocorticoid receptor (GR) is mediated by hormone binding, receptor dimerization, and coactivator recruitment. Here, we report the crystal structure of the human GR ligand binding domain (LBD) bound to dexamethasone and a coactivator motif derived from the transcriptional intermediary factor 2. Despite structural similarity to other steroid receptors, the GR LBD adopts a surprising dimer configuration involving formation of an intermolecular beta sheet. Functional studies demonstrate that the novel dimer interface is important for GR-mediated activation. The structure also reveals an additional charge clamp that determines the binding selectivity of a coactivator and a distinct ligand binding pocket that explains its selectivity for endogenous steroid hormones. These results establish a framework for understanding the roles of protein-hormone and protein-protein interactions in GR signaling pathways.

Asymmetry in the PPARγ/RXRα Crystal Structure Reveals the Molecular Basis of Heterodimerization among Nuclear Receptors

Abstract The nuclear receptor PPARγ/RXRα heterodimer regulates glucose and lipid homeostasis and is the target for the antidiabetic drugs GI262570 and the thiazolidinediones (TZDs). We report the crystal structures of the PPARγ and RXRα LBDs complexed to the RXR ligand 9- cis -retinoic acid (9cRA), the PPARγ agonist rosiglitazone or GI262570, and coactivator peptides. The PPARγ/RXRα heterodimer is asymmetric, with each LBD deviated ∼10° from the C2 symmetry, allowing the PPARγ AF-2 helix to interact with helices 7 and 10 of RXRα. The heterodimer interface is composed of conserved motifs in PPARγ and RXRα that form a coiled coil along helix 10 with additional charge interactions from helices 7 and 9. The structures provide a molecular understanding of the ability of RXR to heterodimerize with many nuclear receptors and of the permissive activation of the PPARγ/RXRα heterodimer by 9cRA.

Crystallization of protein-ligand complexes.

Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals.

Production of monoclonal antibodies using recombinant baculovirus displaying gp64-fusion proteins.

Generation of protein immunogens is often a rate-limiting step in the production of monoclonal antibodies (Mabs). Expressing domains of proteins as fusions to the baculovirus surface glycoprotein gp64 displays foreign proteins on the surface of the virion. Antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the gp64 nucleotide sequence. This method allows immunization with whole virus, eliminating the need for purification of target antigens. Affinity-matured Mabs to the human nuclear receptors LXRbeta and FXR have been produced using baculovirus particles displaying gp64/nuclear receptor fusion proteins as the immunizing agent. Immunizations were performed directly with pelleted virus using the Repetitive Immunization Multiple Sites (RIMMS) immunization strategy for rapid Mab production. All Mabs were identified using insect cells infected with the immunizing virus. Characterization of these antibodies shows them to be class-switched and specific for LXRbeta or FXR. Additionally, high affinity antibodies that recognize gp64 and neutralize baculovirus infection of insect cells were isolated. Use of the recombinant baculovirus gp64 display system makes possible the production of Mabs once a partial DNA sequence is known. This allows the generation of antibodies prior to the isolation of purified protein, in turn providing antibodies to facilitate purification, characterization and immunolocalization of proteins.

Structure and Function of the Glucocorticoid Receptor Ligand Binding Domain

After binding to an activating ligand, such as corticosteroid, the glucocorticoid receptor (GR) performs an impressive array of functions ranging from nuclear translocation, oligomerization, cofactor/kinase/transcription factor association, and DNA binding. One of the central functions of the receptor is to regulate gene expression, an activity triggered by ligand binding. In this role, GR acts as an adapter molecule by encoding the ligands message within the structural flexibility of the ligand binding domain (LBD). The purpose of this review is to discuss the many structural and functional features of the GR LBD in light of recent successful biochemical and crystallographic studies. Progress in this area of research promises to reveal new strategies and insights allowing for the design of novel drugs to treat inflammatory diseases, diabetic conditions, steroid resistance, and cancers.

The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist

The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-like in vitro potency. Its X-ray crystal structure in the GR LBD (Glucocorticoid ligand-binding domain) is described and compared to other reported structures of steroidal GR agonists in the GR LBD (3E7C).

Cloning of the rat and human mitochondrial branched chain aminotransferases (BCATm).

The rat and human mitochondrial branched chain aminotransferase (BCAT(m)) cDNAs have been isolated and shown to encode mature proteins of 41.2 and 41.3 kDa with presequences of 27 amino acids. When rat BCAT(m) is overexpressed in COS-1 cells, the protein exhibits BCAT activity and correct processing of the mitochondrial targeting sequence. Southern blot analysis of genomic DNA from a panel of rodent-human somatic cell hybrids revealed that the human BCAT(m) gene resides on chromosome 19 and the human cytosolic enzyme (BCAT(c)) gene on chromosome 12. Finally, the nomenclature BCAT1 for the cytosolic gene and BCAT2 for the mitochondrial BCAT gene is proposed.

The Role of Phosphodiesterase 12 (PDE12) as a Negative Regulator of the Innate Immune Response and the Discovery of Antiviral Inhibitors

Background: PDE12 degrades 2′,5′-oligoadenylate, a second messenger involved in the antiviral action of interferon. Results: Inactivation of the PDE12 gene and novel inhibitors of the enzyme render cells resistant to more than one virus. Conclusion: PDE12 negatively regulates the innate immune response, and inhibitors of PDE12 have antiviral activity. Significance: PDE12 inhibitors have the potential to be broadly acting antiviral medicines. 2′,5′-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2′,5′-oligoadenylate (2–5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2–5A. Phosphodiesterase 12 (PDE12) was the first cellular 2–5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2–5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2–5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.

Expression, purification, and characterization of multiple, multifunctional human glucocorticoid receptor proteins

The glucocorticoid receptor (GR) is a nuclear receptor protein that plays a central role in glucose homeostasis, the stress response, control of the hypothalamic-pituitary-adrenal axis, and immuno-inflammatory processes via binding of the natural steroid, cortisol. GR is a well-validated drug target and continues to be an important target for new drug discovery efforts. Here, we describe a basic and simple method for Escherichia coli expression and purification of a variety of human GR proteins that contain all three of the functional domains of the protein: the activation function-1 domain, the DNA-binding domain, and the ligand-binding domain. We present characterization data to show that these purified, multifunctional GR proteins are active for ligand, coactivator, and DNA-binding. The work presented here should serve as a reference for future mechanistic, structural and drug discovery efforts that require purified, full or near full length, GR protein.