Sequence annotation of nuclear receptor ligand-binding domains by automated homology modeling

The quality of three-dimensional homology models derived from protein sequences provides an independent measure of the suitability of a protein sequence for a certain fold. We have used automated homology modeling and model assessment tools to identify putative nuclear hormone receptor ligand-binding domains in the genome of Caenorhabditis elegans. Our results indicate that the availability of multiple crystal structures is crucial to obtaining useful models in this receptor family. The majority of annotated mammalian nuclear hormone receptors could be assigned to a ligand-binding domain fold by using the best model derived from any of four template structures. This strategy also assigned the ligand-binding domain fold to a number of C.elegans. sequences without prior annotation. Interestingly, the retinoic acid receptor crystal structure contributed most to the number of sequences that could be assigned to a ligand-binding domain fold. Several causes for this can be suggested, including the high quality of this protein structure in terms of our assessment tools, similarity between the biological function or ligand of this receptor and the modeled genes and gene duplication in C.elegans.     

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.     

Heterodimeric complex of RAR and RXR nuclear receptor ligand-binding domains: purification, crystallization, and preliminary X-ray diffraction analysis

Both the human retinoic acid receptor alpha (hRARalpha) and a constitutively active mutant (F318A) of the mouse retinoid X receptor alpha (mRXR alpha F318A) ligand-binding domains were separately overexpressed in Escherichia coli, copurified as a heterodimer in a two-step procedure, and cocrystallized with an RAR alpha-specific antagonist by using polyethylene glycol 10,000 as precipitant. The crystals grew in the hexagonal space group P6(1)22 displaying the unit cell parameters a = b = 116.6 A and c = 207.8 A. They diffracted X-ray to a limit of 2.2-A resolution. The asymmetric unit comprises one heterodimer and the crystal contains 60% solvent. The structure was determined by molecular replacement and is currently being refined.     

Identification of peptide ligand-binding domains within the human motilin receptor using photoaffinity labeling.

The cDNA encoding the human motilin receptor was recently cloned and found to represent a G protein-coupled receptor that is structurally related to the growth hormone secretagogue receptors. Together, these represent a new Class I receptor family. Our aim in the present work is to gain insight into the molecular basis of binding of motilin to its receptor using photoaffinity labeling. To achieve this, we developed a Chinese hamster ovary cell line that overexpressed functional motilin receptor (CHO-MtlR; 175,000 sites per cell, with K(i) = 2.3 +/- 0.4 nm motilin and EC(50) = 0.3 +/- 0.1 nm motilin) and a radioiodinatable peptide analogue of human motilin that incorporated a photolabile p-benzoyl-l-phenylalanine (Bpa) residue into its pharmacophoric domain. This probe, [Bpa(1),Ile(13)]motilin, was a full agonist at the motilin receptor that increased intracellular calcium in a concentration-dependent manner (EC(50) = 1.5 +/- 0.4 nm). This photolabile ligand bound specifically and with high affinity to the motilin receptor (K(i) = 12.4 +/- 1.0 nm), and covalently labeled that molecule within its M(r) = 45,000 deglycosylated core. Cyanogen bromide cleavage demonstrated its covalent attachment to fragments of the receptor having apparent M(r) = 6,000 and M(r) = 31,000. These were demonstrated to represent fragments that included both the first and the large second extracellular loop domains, with the latter representing a unique structural feature of this receptor. The spatial approximation of the pharmacophoric domain of motilin with these receptor domains support their functional importance as well.     

Over-expression and purification of isotopically labeled recombinant ligand-binding domain of orphan nuclear receptor human B1-binding factor/human liver receptor homologue 1 for NMR studies

The human hepatitis B virus enhancer II B1 binding factor (hB1F), which regulates the expression of hepatitis B virus genes, is identified as a nuclear receptor. It regulates several liver-specific genes and plays an important role in the bile acid biosynthesis pathway. A significantly optimized protocol has been worked out to prepare 15N and/or 13C-labeled hB1F ligand-binding domain in minimal medium with high yields for NMR studies. Under the various conditions optimized for the purification of His6-hB1F ligand-binding domain, the yield of the purified protein is estimated to be 25-30 mg from 0.5 L of M9 minimal media. Electrospray ionization mass spectrometry data confirm the correctness of the primary sequence. Dynamic light scattering experiment proves that the protein exists as a monomeric form. In addition, the circular dichroism results show that the protein has a well-regulated secondary structure and a high alpha-helical content in ammonium bicarbonate buffer at 20 degrees C and pH 7.4. Finally, uniformly 15N-labeled protein is characterized by a TROSY-HSQC spectrum, and the dispersion of 15N-1H cross-peaks in the spectrum indicates the presence of well-ordered and properly folded protein as a monomer.     

A family-based approach reveals the function of residues in the nuclear receptor ligand-binding domain

Literature studies, 3D structure data, and a series of sequence analysis techniques were combined to reveal important residues in the structure and function of the ligand-binding domain of nuclear hormone receptors. A structure-based multiple sequence alignment allowed for the seamless combination of data from many different studies on different receptors into one single functional model. It was recently shown that a combined analysis of sequence entropy and variability can divide residues in five classes; (1) the main function or active site, (2) support for the main function, (3) signal transduction, (4) modulator or ligand binding and (5) the rest. Mutation data extracted from the literature and intermolecular contacts observed in nuclear receptor structures were analyzed in view of this classification and showed that the main function or active site residues of the nuclear receptor ligand-binding domain are involved in cofactor recruitment. Furthermore, the sequence entropy-variability analysis identified the presence of signal transduction residues that are located between the ligand, cofactor and dimer sites, suggesting communication between these regulatory binding sites. Experimental and computational results agreed well for most residues for which mutation data and intermolecular contact data were available. This allows us to predict the role of the residues for which no functional data is available yet. This study illustrates the power of family-based approaches towards the analysis of protein function, and it points out the problems and possibilities presented by the massive amounts of data that are becoming available in the "omics era". The results shed light on the nuclear receptor family that is involved in processes ranging from cancer to infertility, and that is one of the more important targets in the pharmaceutical industry.     

Helix 8 Leu in the CB1 cannabinoid receptor contributes to selective signal transduction mechanisms

The intracellular C-terminal helix 8 (H8) of the CB(1) cannabinoid receptor deviates from the highly conserved NPXXY(X)(5,6)F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB(1) with those of an L7.60F mutation and an L7.60I mutation that mimics the CB(2) sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca(2+) current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Galpha(i3) but not Galpha(0A) in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Galpha(i3). Furthermore, Galpha(i3) but not Galpha(0A) enhanced basal facilitation ratio, suggesting that Galpha(i3) is responsible for CB(1) tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Galpha(i1) or Galpha(i2) but not with Galpha(i3). Molecular dynamics simulations of WT CB(1) receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)(5,6)L contributes to maximal activity of the CB(1) receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.     

Helix 3-helix 5 interactions in steroid hormone receptor function

Steroid hormones working through their receptors regulate a wide variety of physiologic processes necessary for normal homeostasis. Recent years have witnessed great advances in our understanding of how these hormones interact with their receptors, and have brought us closer to the era of directed drug design. We previously described a novel intramolecular interaction between helix 3 and helix 5 which is responsible for a Mendelian form of human hypertension. Further studies revealed that this interaction is highly conserved throughout the steroid hormone receptor family and functions as a key regulator of steroid hormone receptor sensitivity and specificity. Here, we review the contribution of helix 3-helix 5 interaction to steroid hormone receptor activity, with an eye towards how this knowledge may aid in the creation of novel therapeutic agonists and antagonists.     

Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor

The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.     

Extracellular domains of the neurokinin-1 receptor: structural characterization and interactions with substance P

The technical difficulties associated with the structure determination of membrane proteins have limited the structural information available for the ligand binding to G-protein coupled receptors (GPCRs). Here, we describe a reductionist approach to GPCR structure determination in which the extracellular domains of the receptor are examined by high-resolution NMR in the presence of a membrane mimetic. The resulting structural features are then incorporated into a molecular model of the receptor, utilizing the x-ray structure of rhodopsin to generate the topological orientation of the transmembrane helices. The results of our study of the neurokinin-1 receptor (NK-1R) and its interactions with substance P (SP) are detailed here. The structure of the N-terminus, NK-1R(1-39), and of the third extracellular loop, NK-1R(264-290), in the presence of dodecylphosphocholine micelles is described. Our findings provide a structural basis for the interpretation of the results from other methods including mutagenesis, fluorescence, and photoaffinity labeling experiments, resulting in an experimentally based, high-resolution model of SP binding to NK-1R.     

Helix 8 of the viral chemokine receptor ORF74 directs chemokine binding

The constitutively active G-protein-coupled receptor and viral oncogene ORF74, encoded by Kaposi sarcoma-associated herpesvirus (human herpesvirus 8), binds a broad range of chemokines, including CXCL1 (agonist), CXCL8 (neutral ligand), and CXCL10 (inverse agonist). Although chemokines interact with the extracellular N terminus and loops of the receptor, we demonstrate that helix 8 (Hx8) in the intracellular carboxyl tail (C-tail) of ORF74 directs chemokine binding. Partial deletion of the C-tail resulted in a phenotype with reduced constitutive activity but intact regulation by ligands. Complete deletion of the C-tail, including Hx8, resulted in an inactive phenotype that lacks CXCL8 binding sites and has an increased number of binding sites for CXCL10. Similar effects were obtained with the single R7.61(322)W or Q7.62(323)P mutations in Hx8. We propose that the conserved charged or polar side chain at position 7.61 has a specific role in stabilizing the end of transmembrane domain 7 (TM7). Disruption of Hx8 by deletion or mutation distorts an H-bonding network, involving highly conserved amino acids within TM2, TM7, and Hx8, that is crucial for positioning of the TM domains, coupling to Galphaq, and CXCL8 binding. Thus, Hx8 appears to exert a key role in receptor stabilization through the conserved residue R7.61, directing the ligand binding profile of ORF74 and likely also that of other class A G-protein-coupled receptors.     

Local motifs involved in the canonical structure of the ligand-binding domain in the nuclear receptor superfamily

Structural and sequence alignment analyses have revealed the existence of class-dependent and -independent local motifs involved in the overall fold of the ligand-binding domain (LBD) in the nuclear receptor (NR) superfamily. Of these local motifs, three local motifs, i.e., AF-2 fixed motifs, were involved in the agonist conformation of the activation function-2 (AF-2) region of the LBD. Receptor–agonist interactions increased the stability of these AF-2 fixed motifs in the agonist conformation. In contrast, perturbation of the AF-2 fixed motifs by a ligand or another protein molecule led the AF-2 architecture to adopt an antagonist conformation. Knowledge of this process should provide us with novel insights into the ‘agonism’ and ‘antagonism’ of NRs.     

Androgen receptor ligand-binding domain interaction and nuclear receptor specificity of FXXLF and LXXLL motifs as determined by L/F swapping

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.     

Model of the whole rat AT1 receptor and the ligand-binding site

We present a three-dimensional model of the rat type 1 receptor (AT₁) for the hormone angiotensin II (Ang II). Ang II and the AT₁ receptor play a critical role in the cell-signaling process responsible for the actions of renin–angiotensin system in the regulation of blood pressure, water-electrolyte homeostasis and cell growth. Development of improved therapeutics would be significantly enhanced with the availability of a 3D-structure model for the AT₁ receptor and of the binding site for agonists and antagonists. This model was constructed using a combination of computation and homology-modeling techniques starting with the experimentally determined three-dimensional structure of bovine rhodopsin (PDB#1F88) as a template. All 359 residues and two disulfide bonds in the rat AT₁ receptor have been accounted for in this model. Ramachandran-map analysis and a 1 nanosecond molecular dynamics simulation of the solvated receptor with and without the bound ligand, Ang II, lend credence to the validity of the model. Docking calculations were performed with the agonist, Ang II and the antihypertensive antagonist, losartan.      

Analysis of post-translational CCR8 modifications and their influence on receptor activity

Post-translational modifications of the extracellular portions of receptors located in the cell membrane can contribute to modulating their biological activity. Using a mutagenesis approach in which single or multiple Tyr-to-Phe, Thr-to-Ala, Ser-to-Ala, and Asn-to-Gln substitutions were made at the appropriate positions, we analyzed the sulfation and glycosylation state of the murine CCR8 chemokine receptor, and the way in which these post-translational modifications affect CCR8 activity. A Y14Y15-to-F14F15 CCR8 mutant was less sulfated than the wild-type receptor. An N8-to-Q8 mutant was less glycosylated than wild-type, and a double T10T12-to-A10A12 mutant showed even less glycosylation. We established a flow cytometric analysis with an Fc-fused form of mouse CCL1 to determine precisely the ligand-binding activity of these mutants. Single mutants at amino acid positions 8, 10 or 12 bound CCL1-Fc similarly to wild-type CCR8, whereas the F14F15 double mutant was essentially inactive and the A10A12 double mutant showed about 65% of wild-type ligand-binding activity. Calcium flux activity assays were performed with these mutants, yielding results consistent with those from the ligand binding assays. These data indicate that sulfation at specific positions of the N-terminal domain of mouse CCR8 is critical for its biological activity, whereas glycosylation has a minor influence.