Structural and functional characterization of a novel type of ligand-independent RXR-USP receptor

Retinoid X receptor (RXR) and Ultraspiracle (USP) play a central role as ubiquitous heterodimerization partners of many nuclear receptors. While it has long been accepted that a wide range of ligands can activate vertebrate/mollusc RXRs, the existence and necessity of specific endogenous ligands activating RXR-USP in vivo is still matter of intense debate. Here we report the existence of a novel type of RXR-USP with a ligand-independent functional conformation. Our studies involved Tribolium USP (TcUSP) as representative of most arthropod RXR-USPs, with high sequence homology to vertebrate/mollusc RXRs. The crystal structure of the ligand-binding domain of TcUSP was solved in the context of the functional heterodimer with the ecdysone receptor (EcR). While EcR exhibits a canonical ligand-bound conformation, USP adopts an original apo structure. Our functional data demonstrate that TcUSP is a constitutively silent partner of EcR, and that none of the RXR ligands can bind and activate TcUSP. These findings together with a phylogenetic analysis suggest that RXR-USPs have undergone remarkable functional shifts during evolution and give insight into receptor-ligand binding evolution and dynamics.     

Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects

The major postembryonic developmental events happening in insect life, including molting and metamorphosis, are regulated and coordinated temporally by pulses of ecdysone. The biological activity of this steroid hormone is mediated by two nuclear receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein (USP). The crystal structure of the ligand-binding domain from the lepidopteran Heliothis virescens USP reported here shows that the loop connecting helices H1 and H3 precludes the canonical agonist conformation. The key residues that stabilize this unique loop conformation are strictly conserved within the lepidopteran USP family. The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of this receptor provides the template for designing inhibitors, which could be utilized as a novel type of environmentally safe insecticides.     

Thermodynamic characterization of interleukin-8 monomer binding to CXCR1 receptor N-terminal domain

Chemokines elicit their function by binding receptors of the G-protein-coupled receptor class, and the N-terminal domain (N-domain) of the receptor is one of the two critical ligand-binding sites. In this study, the thermodynamic basis for binding of the chemokine interleukin-8 (IL-8) to the N-domain of its receptor CXCR1 was characterized using isothermal titration calorimetry. We have shown previously that only the monomer of IL-8, and not the dimer, functions as a high-affinity ligand, so in this study we used the IL-8(1-66) deletion mutant which exists as a monomer. Calorimetry data indicate that the binding is enthalpically favored and entropically disfavored, and a negative heat capacity change indicates burial of hydrophobic residues in the complex. A characteristic feature of chemokine receptor N-domains is the large number of acidic residues, and experiments using different buffers show no net proton transfer, indicating that the CXCR1 N-domain acidic residues are not protonated in the binding process. CXCR1 N-domain peptide is unstructured in the free form but adopts a more defined structure in the bound form, and so binding is coupled to induction of the structure of the N-domain. Measurements in the presence of the osmolyte, trimethylamine N-oxide, which induces the structure of unfolded proteins, show that formation of the coupled N-domain structure involves only small DeltaH and DeltaS changes. These results together indicate that the binding is driven by packing interactions in the complex that are enthalpically favored, and are consistent with the observation that the N-domain binds in an extended form and interacts with multiple IL-8 N-loop residues over a large surface area.     

Characterization of ligand-binding properties of the human BMP type II receptor extracellular domain

ActR-IIA, ActR-IIB, and BMPR-II are low-affinity type II receptors that bind bone morphogenetic proteins (BMPs) in the same overall manner. The binding of BMPs by ActR-IIs has been analyzed structurally and functionally, but no detailed analysis of BMPR-II has been reported. The objective of this study was to determine ligand-binding epitopes and specificity determinants in two regions, the hydrophobic patch and the A-loop of the BMPR-II extracellular domain (ECD). A series of alanine-substituted variants was generated using a recently published X-ray structure of the unliganded form of the ovine BMPR-II ECD as a guide. These variants were characterized using one-dimensional NMR and functional activity assays with BMP-2, BMP-7 and GDF-5 as ligands. The results showed that alanine substitutions of conserved residues W85 and Y113 within the hydrophobic patch of the ECD differentially perturbed BMP ligand binding without disrupting receptor folding, suggesting that they are critical determinants for ligand binding and ligand specificity. Our results further revealed that the nonconserved residue L69 in the hydrophobic patch contributes to ligand-binding activity and specificity. Mutations of several residues within the A-loop resulted in minimal effects on the binding of the different BMP ligands. Overall, these observations identify several amino acid residues that play different roles in BMPR-II and ActR-II and thereby enable BMPR-II and ActR-IIs to bind different subclasses of BMP ligands.     

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.     

Using nondenaturing mass spectrometry to detect fortuitous ligands in orphan nuclear receptors

Nondenaturing electrospray mass spectrometry (ESI-MS) has been used to reveal the presence of potential ligands in the ligand-binding domain (LBD) of orphan nuclear receptors. This new approach, based on supramolecular mass spectrometry, allowed the detection and identification of fortuitous ligands for the retinoic acid-related orphan receptor beta (RORbeta) and the ultraspiracle protein (USP). These fortuitous ligands were specifically captured from the host cell with the proper stoichiometry. After organic extraction, these molecules have been characterized by classic analytical methods and identified as stearic acid for RORbeta and a phosphatidylethanolamine (PE) for USP, as confirmed by crystallography. These molecules act as "fillers" and may not be the physiological ligands, but they prove to be essential to stabilize the active conformation of the LBD, enabling its crystallization. The resulting crystal structures provide a detailed picture of the ligand-binding pocket, allowing the design of highly specific synthetic ligands that can be used to characterize the function of orphan nuclear receptors. An additional advantage of this new method is that it is not based on a functional test and that it can detect low-affinity ligands.     

Dermenkephalin and deltorphin I reveal similarities within ligand-binding domains of mu- and delta-opioid receptors and an additional address subsite on the delta-receptor.

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to the mu- and delta-opioid receptors, respectively. The amino-terminal domains Tyr-D-X-Phe (where X is either Ala or Met) of these peptides behave as selective and potent mu-receptor ligands. Routing of Tyr-D-X-Phe to the delta- or the mu- receptor is associated with the presence or the absence at the C-terminus of an additional hydrophobic and negatively charged tetrapeptide by-passing the mu-addressing ability of the amino-terminal moiety. A study of 20 Tyr-D-X-Phe-Y-NH2 analogs with substitution of X and Y by neutral, hydrophobic, aromatic amino acids as well as by charged amino acid residues shows that tetrapeptides maintain high binding affinity and selectivity for the mu-opioid receptor. Although residue in position 4 serves a delta-address function, the tripeptide motif at the C-terminus of dermenkephalin and deltorphin I are critical components for high selectivity at delta-opioid receptor. Results demonstrate that mu- and delta-opioid receptors share topologically equivalent ligand-binding domains, or ligand-binding sequences similarities, that recognized Tyr-D-X-Phe as a consensus message-binding sequence. The delta-receptor additionally contains a unique address subsite at or near the conserved binding domain that accommodates the C-terminal tetrapeptide motif of dermenkephalin and deltorphin I.     

The role of tyrosine at the ligand-binding site of the nicotinic acetylcholine receptor

Identification of the critical residues in a receptor's ligand-binding site provides valuable structural information important for understanding the basis for ligand recognition. The design of specific ligands targeted for receptor action will depend to a great extent on detailed structural knowledge of this kind. Although the nicotinic acetylcholine receptor (nAChR) is perhaps the best characterized of all receptors, the detailed configuration of the ligand-binding site remains unknown. Structural comparisons of nicotinic agonists and antagonists have long predicted a negative subsite on the receptor to interact with the positively charged alkyl-ammonium moiety common to nearly all nicotinic agents. We have used intrinsic fluorescence spectroscopic analyses together with binding studies of selectively modified peptide fragments of the nAChR to suggest that one or two invariant tyrosine residues at positions 190 and 198 on the alpha-subunit provide the critical negative subsite required for ligand binding. Tyrosines may similarly be part of the negative subsite of muscarinic receptors and other neurotransmitter receptors that bind cationic ligands.     

Crystal structure of recombinant murine adipocyte lipid-binding protein.

Adipocyte lipid-binding protein (ALBP) is the adipocyte member of an intracellular hydrophobic ligand-binding protein family. ALBP is phosphorylated by the insulin receptor kinase upon insulin stimulation. The crystal structure of recombinant murine ALBP has been determined and refined to 2.5 A. The final R factor for the model is 0.18 with good canonical properties. Crystalline ALBP has a conformation which is essentially identical to that of intestinal fatty acid binding protein and myelin P2 protein. Although the crystal structure is of the apo- form, a cavity resembling that in other family members is present. It contains a number of bound and implied unbound water molecules and shows no large obvious portal to the external milieu. The cavity of ALBP, which by homology is the ligand-binding site, is formed by both polar and hydrophobic residues among which is tyrosine 19. Y19 is phosphorylated by the insulin receptor kinase as described in the accompanying paper [Buelt, M. K., Xu, Z., Banaszak, L. J., & Bernlohr, D. A. (1992) Biochemistry (following paper in this issue)]. By comparing ALBP with the earlier structural results on intestinal fatty acid binding protein, it is now possible to delineate conserved amino acids which help form the binding site in this family.     

Dual role of the Jak1 FERM and kinase domains in cytokine receptor binding and in stimulation-dependent Jak activation

Jak1 is a tyrosine kinase that noncovalently forms tight complexes with a variety of cytokine receptors and is critically involved in signal transduction via cytokines. Jaks are predicted to have a 4.1, ezrin, radixin, moesin (FERM) domain at their N terminus. FERM domains are composed of three structurally unrelated subdomains (F1, F2, and F3) which are in close contact to one another and form the clover-shaped FERM domain. We generated a model structure of the Jak1 FERM domain, based on solved FERM structures and the alignments with other FERM domains. To destabilize different subdomains and to uncover their exact function, we mutated specific hydrophobic residues conserved in FERM domains and involved in hydrophobic core interactions. In this study, we show that the structural integrity of the F2 subdomain of the FERM domain of Jak1 is necessary to bind the IFN-gammaRalpha. By mutagenesis of hydrophobic residues in the hydrophobic core between the three FERM subdomains, we find that the structural context of the FERM domain is necessary for the inhibition of Jak1 phosphorylation. Thus, FERM domain mutations can have repercussions on Jak1 function. Interestingly, a mutation in the kinase domain (Jak1-K907E), known to abolish the catalytic activity, also leads to an impaired binding to the IFN-gammaRalpha when this mutant is expressed at endogenous levels in U4C cells. Our data show that the structural integrity of both the FERM domain and of the kinase domain is essential for both receptor binding and catalytic function/autoinhibition.     

Correlating efficacy and desensitization with GluK2 ligand-binding domain movements

Gating of AMPA- and kainate-selective ionotropic glutamate receptors can be defined in terms of ligand affinity, efficacy and the rate and extent of desensitization. Crucial insights into all three elements have come from structural studies of the ligand-binding domain (LBD). In particular, binding-cleft closure is associated with efficacy, whereas dissociation of the dimer formed by neighbouring LBDs is linked with desensitization. We have explored these relationships in the kainate-selective subunit GluK2 by studying the effects of mutating two residues (K531 and R775) that form key contacts within the LBD dimer interface, but whose truncation unexpectedly attenuates desensitization. One mutation (K531A) also switches the relative efficacies of glutamate and kainate. LBD crystal structures incorporating these mutations revealed several conformational changes that together explain their phenotypes. K531 truncation results in new dimer contacts, consistent with slower desensitization and sideways movement in the ligand-binding cleft correlating with efficacy. The tested mutants also disrupted anion binding; no chloride was detected in the dimer-interface site, including in R775A where absence of chloride was the only structural change evident. From this, we propose that the charge balance in the GluK2 LBD dimer interface maintains a degree of instability, necessary for rapid and complete desensitization.     

Ligand specificity determined by differentially arranged common ligand-binding residues in bacterial amino acid chemoreceptors Tsr and Tar

Escherichia coli has closely related amino acid chemoreceptors with distinct ligand specificity, Tar for l-aspartate and Tsr for l-serine. Crystallography of the ligand-binding domain of Tar identified the residues interacting with aspartate, most of which are conserved in Tsr. However, swapping of the nonconserved residues between Tsr and Tar did not change ligand specificity. Analyses with chimeric receptors led us to hypothesize that distinct three-dimensional arrangements of the conserved ligand-binding residues are responsible for ligand specificity. To test this hypothesis, the structures of the apo- and serine-binding forms of the ligand-binding domain of Tsr were determined at 1.95 and 2.5 Å resolutions, respectively. Some of the Tsr residues are arranged differently from the corresponding aspartate-binding residues of Tar to form a high affinity serine-binding pocket. The ligand-binding pocket of Tsr was surrounded by negatively charged residues, which presumably exclude negatively charged aspartate molecules. We propose that all these Tsr- and Tar-specific features contribute to specific recognition of serine and aspartate with the arrangement of the side chain of residue 68 (Asn in Tsr and Ser in Tar) being the most critical.