Chemical and biochemical issues related to X-ray crystallography of the ligand-binding domain of estrogen receptor alpha

Careful attention to technical issues preceded successful crystallography of the ligand-binding domain of estrogen receptor alpha (ERalpha) complexed with CP-336156, a nonsteroidal estrogen agonist/antagonist. An affinity column based on immobilized estradiol was prepared according to the scheme of Greene et al. (Greene, G. L., Nolan, C., Engler, J. P., and Jensen, E. V. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5115-5119). It was shown by X-ray crystallography that the major and less polar isomer of the affinity column precursor was 17alpha-((S)-2',3'-epoxyprop-1'-yl)estra-1,3,5(10)-triene-3,17beta-diol. This diastereomer was coupled to Thiopropyl Sepharose, with coupling monitored by observing loss of the phenolic absorption band of estradiol from the reaction supernatant, and gave an affinity matrix containing about 9 micromol of estradiol per milliliter of wet gel. Recombinant ERalpha ligand binding domain was selectively removed from E. coli cell lysate by binding to the column and was partly S-carboxymethylated by treatment with iodoacetic acid while bound to the column as described by previous workers. After being eluted from the column as a complex with drug, the receptor fragment was shown by mass spectrometry to be a mixture of differently modified forms. It was further S-carboxymethylated in solution, after which anion-exchange chromatography was used to isolate protein in which two of the four cysteine residues were S-carboxymethylated. This material, which afforded diffraction-quality crystals, was subjected to digestion with trypsin and peptide mapping analysis by HPLC coupled with mass spectrometry. For this experiment, the two previously unmodified cysteines were alkylated with 4-vinylpyridine to allow definitive identification. It was shown that Cys-417 and Cys-530 were S-carboxymethylated in the crystallized protein, while Cys-381 and Cys-447 remained unmodified. Close attention to such technical issues may be important in structural studies of other nuclear receptors, a very important class of potential drug targets.     

The human estrogen receptor hormone binding domain dimerizes independently of ligand activation

High level expression of biochemically active human estrogen receptor hormone binding domain (hER-HBD) was achieved using a Saccharomyces cerevisae expression system. Using dissociation kinetic analysis, density gradient centrifugation and cross-linking studies, a spontaneous dimerization activity of hER-HBD independent of the presence of the DNA binding domain, ligand, and of elevated temperature is demonstrated.     

Identification of covalent attachment site of antiestrogenic estradiol 11 beta-derivatives on human estrogen receptor alpha ligand-binding domain

Affinity labeling of human estrogen receptor alpha (ERalpha) by high affinity and antiestrogenic estradiol (E(2)) 11 beta-derivatives, 11 beta-bromoacetamidoethoxyphenylE(2) (11BAEOPE(2)) and 11 beta-bromoacetamidopentoxyphenylE(2) (11BAPOPE(2)) was studied using glutathione-S-transferase (GST) fused to the ligand-binding domain (LBD) of human ERalpha. To identify and quantify the electrophile covalent attachment sites on LBD, [(14)C]11BAEOPE(2)- and [(14)C]11BAPOPE(2)-alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE(2) and 11BAPOPE(2), respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys(530)Lys(531) LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the covalent attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD-ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys(530) could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common attachment to Cys(530) of estrogenic E(2) 17 alpha-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor alpha, J. Steroid Biochem. Mol. Biol. 98 (4-5), in press] and antiestrogenic E(2) 11 beta-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.     

The nicotinic receptor ligand binding domain

The ligand binding domain (LBD) of the nicotinic acetylcholine receptor has served as a prototype for understanding molecular recognition in the family of neurotransmitter-gated ion channels. During the past fifty years, studies progressed from fundamental electrophysiological analyses of ACh-evoked ion flow, to biochemical purification of the receptor protein, pharmacological measurements of ligand binding, molecular cloning of receptor subunits, site-directed mutagenesis combined with functional analysis and recently, atomic structural determination. The emerging picture of the nicotinic receptor LBD is a specialized pocket of aromatic and hydrophobic residues formed at interfaces between protein subunits that changes conformation to convert agonist binding into gating of an intrinsic ion channel.     

The use of in vitro peptide binding profiles and in silico ligand-receptor interaction profiles to describe ligand-induced conformations of the retinoid X receptor alpha ligand-binding domain

It is hypothesized that different ligand-induced conformational changes can explain the different interactions of nuclear receptors with regulatory proteins, resulting in specific biological activities. Understanding the mechanism of how ligands regulate cofactor interaction facilitates drug design. To investigate these ligand-induced conformational changes at the surface of proteins, we performed a time-resolved fluorescence resonance energy transfer assay with 52 different cofactor peptides measuring the ligand-induced cofactor recruitment to the retinoid X receptor-alpha (RXRalpha) in the presence of 11 compounds. Simultaneously we analyzed the binding modes of these compounds by molecular docking. An automated method converted the complex three-dimensional data of ligand-protein interactions into two-dimensional fingerprints, the so-called ligand-receptor interaction profiles. For a subset of compounds the conformational changes at the surface, as measured by peptide recruitment, correlate well with the calculated binding modes, suggesting that clustering of ligand-receptor interaction profiles is a very useful tool to discriminate compounds that may induce different conformations and possibly different effects in a cellular environment. In addition, we successfully combined ligand-receptor interaction profiles and peptide recruitment data to reveal structural elements that are possibly involved in the ligand-induced conformations. Interestingly, we could predict a possible binding mode of LG100754, a homodimer antagonist that showed no effect on peptide recruitment. Finally, the extensive analysis of the peptide recruitment profiles provided novel insight in the potential cellular effect of the compound; for the first time, we showed that in addition to the induction of coactivator peptide binding, all well-known RXRalpha agonists also induce binding of corepressor peptides to RXRalpha.     

The steroid binding domain of porcine estrogen receptor

For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), we have made use of affinity labeling of partially purified ER with [3H]tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or alpha-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.     

Curariform antagonists bind in different orientations to the nicotinic receptor ligand binding domain

Curariform alkaloids competitively inhibit muscle acetylcholine receptors (AChR) by bridging the alpha and non-alpha subunits that form the ligand-binding site. Here we delineate bound orientations of d-tubocurarine (d-TC) and its methylated derivative metocurine using mutagenesis, ligand binding measurements, and computational methods. When tested against a series of lysine mutations in the epsilon subunit, the two antagonists show marked differences in the consequences of the mutations on binding affinity. The mutations epsilon L117K, epsilon Y111K, and epsilon L109K decrease affinity of metocurine by up to 3 orders of magnitude but only slightly alter affinity of d-TC. At the alpha subunit face of the binding site, the mutation alpha Y198T decreases affinity of both antagonists, but alpha Y198F preferentially enhances affinity of d-TC. Computation of antagonist docking orientations, based on our structural model of the alpha-epsilon site of the human AChR, indicates distinct orientations of each antagonist; the flatter metocurine fits into a pocket formed principally by the epsilon subunit, whereas the more compact d-TC spans the narrower crevasse between alpha and epsilon subunits. The side chains of epsilon Tyr-111 and epsilon Thr-117 juxtapose one of two quaternary nitrogens in metocurine but are remote from the equivalent quaternary nitrogen in d-TC, which instead closely approaches alpha Tyr-198. The different docked orientations arise through tilt of the curariform scaffold by approximately 60 degrees normal to the nitrogen-nitrogen axis, together with a 20 degrees rotation about the axis. The overall mutagenesis and computational results show that despite their similar structures, d-TC and metocurine bind in distinctly different orientations to the adult human AChR.     

The ligand binding site of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit

The extreme carboxyl-terminal amino acid sequence of the gamma chain of fibrinogen is involved in the binding of this adhesive protein to the platelet integrin glycoprotein (GP) IIb-IIIa, and synthetic peptides corresponding to this region inhibit fibrinogen as well as fibronectin and von Willebrand factor binding to platelets. A chemical cross-linking approach was used to characterize the interaction of a 16-amino acid fibrinogen gamma chain peptide with platelets and to localize the site of its binding to GPIIb-IIIa. This peptide became specifically cross-linked to GPIIb, and platelet stimulation selectively enhanced its cross-linking to this alpha subunit. The cross-linking reaction was specifically inhibited by fibrinogen and an Arg-Gly-Asp peptide but not by an unrelated protein or a substituted peptide. Utilizing a combination of immunochemical mapping, enzymatic and chemical digestions, and amino acid sequencing, the cross-linking site of the gamma chain peptide in GPIIb was localized to a stretch of 21 amino acids. The identified region, GPIIb 294-314, contains the second putative calcium binding domain within GPIIb. The primary structure of this region is highly conserved among alpha subunits of other integrin adhesion receptors. These results identify a discrete region of GPIIb that resides in close proximity to a ligand binding site within GPIIb-IIIa. The homologous region may be involved in the functions of other integrin receptors.     

Control of estrogen receptor ligand binding by Hsp90

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.     

The structure of the interleukin-15 alpha receptor and its implications for ligand binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.     

Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations

Eph receptors and their ephrin ligands are important mediators of cell-cell communication. They are divided in two subclasses based on their affinities for each other and on sequence conservation. Receptor-ligand binding within each subclass is fairly promiscuous, while binding cross the subclasses happens rarely. EphA4 is an exception to this general rule, since it has long been known to bind both A- and B-class ephrin ligands but the reason for this exceptional behavior has not been worked out at molecular level. Recent structural and biochemical studies on EphA4 ligand-binding domain alone and in complex with its ligands have addressed this question. However, the published structures of EphA4/ephrin complexes differ considerably from each other and strikingly different explanations for the exceptional promiscuity of EphA4 were proposed. To address these contradictory findings, we have determined a crystal structure of the EphA4 ligand-binding domain at 2.3 Å resolution and show that the receptor has an unprecedented ability to exist in two very different, well-ordered conformations even in the unbound state. Our results suggest that the ligand promiscuity of the Ephs is directly correlated with the structural flexibility of the ligand-binding surface of the receptor.