Thomas G. Consler

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.

Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres.

BACKGROUND We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins. METHODS In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence. RESULTS We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen). CONCLUSIONS This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding.

Product Release Is the Major Contributor tok cat for the Hepatitis C Virus Helicase-catalyzed Strand Separation of Short Duplex DNA

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3′ single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5′-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3′-fluorescein-tagged 21-mer (F21).k cat for this reaction was 0.12 s−1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs (∼2) were disrupted concertedly. The maximal value of the rate constant (k eff) describing the late phase of the reaction (strand separation) was 0.5 s−1, which was 4-fold greater than k cat. Release of HF31 from E·HF31 in the presence of ATP (0.21 s−1) was the major contributor tok cat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.

The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined alpha-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

The formation of a covalent complex between a dipeptide ligand and the src SH2 domain.

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.

Effect of microsphere binding site density on the apparent affinity of an interaction partner

Flow cytometric microsphere‐based binding assays can be used to measure molecular interactions with high sensitivity. We have used multiplexed microsphere technology to explore the effect that binding site density has on the apparent affinity of a soluble interaction partner.

A strategy for predicting the ligand binding competence of recombinant orphan nuclear receptors using biophysical characterization

Publisher Summary Nuclear receptors have been historically associated with the steroid hormone receptors, for example, estrogen and glucocorticoid receptors, by virtue of DNA binding domain sequence homology comprising two zinc finger motifs. Many of these are orphan receptors, having no defined ligand. The nuclear receptors present tempting targets in the pursuit of a systems based research approach as so many at present have been cloned. However, when recombinant forms of a receptor are available before its cognate ligand has been identified, confusion arises on weather an orphan receptor is active for use in vitro assays. Researchers now have access to unparalleled amounts of DNA sequence and genetic data. Families of homologous gene products can be studied with the intent of connecting specific proteins to various disease conditions. Specifically, to apply this type of strategy in studies, the problem has been approached with two premises in the chapter. First, the recombinant constructs of orphan nuclear receptors are engineered to contain domains with hypothetical functional homology to receptors with known activities and ligands. In particular, a lot of knowledge has been gained concerning retinoid X receptor a (RXRα) and the domains necessary for DNA binding, retinoid binding, and self/hetero-association. For the purpose of this study, PPARα, PPARδ, PPARγ, RXRα, and LXRα constructs were created to contain the putative ligand binding domains (LBD). The amino acid residues within this conserved contiguous region have been shown to be both necessary and sufficient to demonstrate ligand binding competence for RXRα and other nuclear receptors. Structurally, the LBDs are composed primarily of multiple α-helicies. Second, each nuclear receptor is characterized using a variety of biophysical techniques. This chapter has presented data that suggests that the usefulness of a recombinant protein can be determined before an appropriate ligand is available. These results are an encouraging start in the attempt to predict the binding competency of recombinant orphan nuclear receptors.