孤儿核受体hB1F／hLRH-1铰链区一个保守结构域对其转录功能的抑制作用

孤儿核受体hB1F(NR5A2 ,也称之为LRH 1、CPF或FTF)在胆汁酸合成代谢、乙肝病毒基因和部分肝特异性基因表达的调控中起着重要的作用。为理解hB1F激活转录的分子机制 ,对其铰链区潜在的功能结构域进行了分析。利用GAL4 DBD融合的hB1F缺失片段所进行的报告基因分析 ,发现了一个位于铰链区的强烈抑制hB1F反式激活能力的结构域。该结构域核心残基的定点突变导致了hB1F反式激活能力的显著上升 ,而且明显地增强hB1F对乙肝病毒增强子II 核心启动子的转录激活能力。生物信息学分析显示该结构域不存在明显的二级结构 ,但有意思的是 ,其氨基酸序列在核受体FTZ F1亚家族的成员中高度保守 ,且不见于其他蛋白质中。转染分析发现 ,该结构域的抑制活性存在于测试的五个不同细胞株中 ,但抑制的强度表现出明显差异。半定量RT PCR分析表明 ,与SF 1不同 ,该结构域抑制转录活性的强度与潜在的结合因子DP10 3的表达水平之间没有相关性。共转染实验还表明 ,参与hB1F转录活性调控的辅激活子SRC 1和辅抑制子SMRT与该抑制作用不直接相关。实验结果提示 ,孤儿核受体hB1F转录活性可能存在一种新的调控机制。     

Backbone <Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments of the ligand binding domain of the human wildtype glucocorticoid receptor and the F602S mutant variant

The glucocorticoid receptor (GR) is a nuclear hormone receptor that regulates key genes controlling development, metabolism, and the immune response. GR agonists are efficacious for treatment of inflammatory, allergic, and immunological disorders. Steroid hormone binding to the ligand-binding domain (LBD) of GR is known to change the structural and dynamical properties of the receptor, which in turn control its interactions with DNA and various co-regulators and drive the pharmacological response. Previous biophysical studies of the GR LBD have required the use of mutant forms to overcome issues with limited protein stability and high aggregation propensity. However, these mutant variants are known to also influence the functional response of the receptor. Here we report a successful protocol for protein expression, purification, and NMR characterization of the wildtype human GR LBD. We achieved chemical shift assignments for 90% of the LBD backbone resonances, with 216 out of 240 non-proline residues assigned in the ¹H–¹⁵N TROSY spectrum. These advancements form the basis for future investigations of allosteric effects in GR signaling.     

Crystal structure of the human receptor activity-modifying protein 1 extracellular domain

Receptor activity-modifying protein (RAMP) 1 forms a heterodimer with calcitonin receptor-like receptor (CRLR) and regulates its transport to the cell surface. The CRLR.RAMP1 heterodimer functions as a specific receptor for calcitonin gene-related peptide (CGRP). Here, we report the crystal structure of the human RAMP1 extracellular domain. The RAMP1 structure is a three-helix bundle that is stabilized by three disulfide bonds. The RAMP1 residues important for cell-surface expression of the CRLR.RAMP1 heterodimer are clustered to form a hydrophobic patch on the molecular surface. The hydrophobic patch is located near the tryptophan residue essential for binding of the CGRP antagonist, BIBN4096BS. These results suggest that the hydrophobic patch participates in the interaction with CRLR and the formation of the ligand-binding pocket when it forms the CRLR.RAMP1 heterodimer.     

Crystal structure of the carbohydrate recognition domain of the H1 subunit of the asialoglycoprotein receptor

The human asialoglycoprotein receptor (ASGPR), also called hepatic lectin, is an integral membrane protein and is responsible for the clearance of desialylated, galactose-terminal glycoproteins from the circulation by receptor-mediated endocytosis. It can be subdivided into four functional domains: the cytosolic domain, the transmembrane domain, the stalk and the carbohydrate recognition domain (CRD). The galactose-binding domains belong to the superfamily of C-type (calcium-dependent) lectins, in particular to the long-form subfamily with three conserved intramolecular disulphide bonds. It is able to bind terminal non-reducing galactose residues and N-acetyl-galactosamine residues of desialated tri or tetra-antennary N-linked glycans. The ASGPR is a potential liver-specific receptor for hepatitis B virus and Marburg virus and has been used to target exogenous molecules specifically to hepatocytes for diagnostic and therapeutic purposes.Here, we present the X-ray crystal structure of the carbohydrate recognition domain of the major subunit H1 at 2.3 A resolution. While the overall fold of this and other known C-type lectin structures are well conserved, the positions of the bound calcium ions are not, indicating that the fold is stabilised by alternative mechanisms in different branches of the C-type lectin family. It is the first CRD structure where three calcium ions form an intergral part of the structure. In addition, the structure provides direct confirmation for the conversion of the ligand-binding site of the mannose-binding protein to an asialoglycoprotein receptor-like specificity suggested by Drickamer and colleagues. In agreement with the prediction that the coiled-coil domain of the ASGPR is separated from the CRD and its N-terminal disulphide bridge by several residues, these residues are indeed not alpha-helical, while in tetranectin they form an alpha-helical coiled-coil.     

Crystal structure of human ISG15 protein in complex with influenza B virus NS1B

ISG15 (interferon-stimulated gene 15), the first ubiquitin-like protein (UBL) identified, has emerged as an important cellular antiviral factor. It consists of two UBL domains with a short linker between them. The covalent attachment of ISG15 to host and viral proteins to modify their functions, similar to ubiquitylation, is named ISGylation. Influenza B virus NS1B protein antagonizes human but not mouse ISGylation because NS1B exhibits species specificity; it only binds human and non-human primate ISG15. Previous studies have demonstrated that the N-terminal UBL domain and linker of ISG15 are required for the binding by NS1B and that the linker plays a large role in the species specificity, but the structural basis for them has not been elucidated. Here we report the crystal structure of human ISG15 in complex with NS1B at a resolution of 2.0 Å. A loop in the ISG15 N-terminal UBL domain inserts into a pocket in the NS1B dimer, forming a high affinity binding site. The nonspecific van der Waals contacts around the ISG15 linker form a low affinity site for NS1B binding. However, sequence alignment reveals that residues in the high affinity site are highly conserved in primate and non-primate ISG15. We propose that the low affinity binding around the ISG15 linker is important for the initial contact with NS1B and that the stable complex formation is largely contributed by the following high affinity interactions between ISG15 N-terminal UBL domain and NS1B. This provides a structural basis for the species-specific binding of ISG15 by the NS1B protein.     

Genomic organization of the human retinoic acid receptor beta 2.

Recently three isoforms of the mouse retinoic acid receptor (mRAR beta 1, mRAR beta 2, mRAR beta 3) have been described, generated from the same gene (Zelent et al., 1991). The isoforms differ in their 5'-untranslated (5'-UTR) and A region, but have identical B to F regions. The N-terminal variability of mRAR beta 1/beta 3 is encoded in the first two exons (E1 and E2), while exon E3 includes N-terminal sequences of the mRAR beta 2 isoform. We have determined the structure of the human RAR beta 2 gene, using a genomic library from K562 cells. The open reading frame is split into eight exons: E3 contains sequences for the N-terminal A region and E4 to E10 encode the common part of the receptor, including the DNA-binding domain and ligand-binding domain. Corresponding to other nuclear receptors, both 'zinc-fingers' of the DNA-binding domain are encoded separately in two exons and the ligand-binding domain is assembled from five exons.     

Structural mobility of the extracellular ligand-binding core of an ionotropic glutamate receptor. Analysis of NMR relaxation dynamics

Ionotropic glutamate receptors play important roles in a variety of neuronal processes and have been implicated in multiple neurodegenerative diseases. The extracellular ligand-binding (S1S2) core of the GluR2 subtype can be expressed in bacteria as a soluble, monomeric protein with binding properties essentially identical to those of the intact receptor. The crystal structure of this protein has been determined in the presence and absence of various agonists and antagonists [Armstrong, N., Sun, Y., Chen, G. Q., and Gouaux, E. (1998) Nature 395, 913-917; Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181]. The protein consists of two lobes, with the S1 segment composing the majority of lobe 1 and the S2 segment composing most of lobe 2. A domain closure upon ligand binding has been postulated, but details of intradomain motions have not been investigated. In this paper, the backbone motions of the ligand-binding core of GluR2 bound to glutamate were studied using (15)N longitudinal (T1) and transverse (T2) relaxation measurements as well as [1H]-15N nuclear Overhauser effects at 500 and 600 MHz. Residues in the agonist-binding pocket exhibited two main classes of motion. Those contacting the alpha-substituents of the ligand glutamate exhibited minimal internal motion, while those contacting the gamma-constituents exhibited exchange dynamics, indicating two dynamically distinct portions of the binding pocket. Also, two residues in transdomain linkers between lobes 1 and 2 show exchange, lending new insight into the previously proposed domain closure hypothesis. Finally, concerted motion of helix F suggests a pathway for ligand dissociation without the necessity of domain reopening.     

Characterization of transactivational property and coactivator mediation of rat mineralocorticoid receptor activation function-1 (AF-1)

The autonomous activation function-2 (AF-2) in the mineralocorticoid receptor (MR) E/F domain is known to play a major role in the ligand-induced transactivation function of MR; however, it remained unclear about the transactivation function of its A/B domain. We therefore tried to characterize the MR A/B domain as the AF-1 and further studied the actions of known coactivators for AF-2 in the E/F ligand-binding domain in the function of the MR A/B domain. Deletion analyses of rat and human MRs revealed that the A/B domains harbor a transactivation function acting as AF-1. The MR mutant (E959Q) with a point mutation in helix 12, which causes a complete loss of MR AF-2 activity, still retained ligand-induced transactivation function, indicating a significant role for AF-1 in the full activity of the ligand-induced MR function. Among the coactivators tested to potentiate the MR AF-2, TIF2 and p300 potentiated the MR AF-1 through two different core regions [amino acids (a.a.) 1-169, a.a. 451-603] and exhibited functional interactions with the MR A/B domain in the cultured cells. However, such interactions were undetectable in a yeast and in an in vitro glutathione-S-transferase pull-down assay, indicating that the functional interaction of TIF2 and p300 with the MR A/B domain to support the MR AF-1 activity require some unknown nuclear factor(s) or a proper modification of the A/B domain in the cells.     

Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.     

1H, 13C, and 15N resonance assignment of a 179 residue fragment of hepatitis C virus non-structural protein 5A

Non-structural protein 5A (NS5A) plays an important role in the life cycle of hepatitis C virus. This proline-rich phosphoprotein is organized into three domains. Besides its role in virus replication and virus assembly, NS5A is involved in a variety of cellular regulation processes. Recent studies on domain 2 and 3 revealed that both belong to the class of intrinsically disordered proteins as they adopt a natively unfolded state. In particular, domain 2 together with its vicinal regions is responsible for NS5A’s multiple interactions with other proteins necessary for virus persistence. The low chemical shift dispersion observed for instrinsically disordered proteins presents a challenge for NMR spectroscopy. Here we report sequential resonance assignment of a 179-residue fragment of NS5A, comprising the entire domain 2, using a set of sensitivity and resolution optimized 3D correlation experiments, as well as amino-acid-type editing in ¹H-¹⁵N correlation spectra. Our assignment reveals the presence of several segments with high propensity to form α-helical structure that may be of importance to the function of this protein fragment as a versatile interaction platform.     

Human genetic polymorphisms in T1R1 and T1R3 taste receptor subunits affect their function

Umami is the typical taste induced by monosodium glutamate (MSG), which is thought to be detected by the heterodimeric G protein-coupled receptor, T1R1 and T1R3. Previously, we showed that MSG detection thresholds differ substantially between individuals and we further showed that nontaster and hypotaster subjects are associated with nonsynonymous single polymorphisms occurring in the T1R1 and T1R3 genes. Here, we show using functional expression that both amino acid substitutions (A110V and R507Q) in the N-terminal ligand-binding domain of T1R1 and the 2 other ones (F749S and R757C), located in the transmembrane domain of T1R3, severely impair in vitro T1R1/T1R3 response to MSG. A molecular model of the ligand-binding region of T1R1/T1R3 provides a mechanistic explanation supporting functional expression data. The data presented here support causal relations between the genotype and previous in vivo psychophysical studies in human evaluating sensitivity to MSG.     

Over-expression and refolding of isotopically labeled recombinant catalytic domain of human macrophage elastase (MMP-12) for NMR studies

Human macrophage elastase (MMP-12) plays an important role in inflammatory processes and is involved in a number of physiological or pathological situations, such as conversion of plasminogen into angiostatin, allergic airway inflammation, vascular remodeling or alteration, as well as emphysema, and has been justified as a novel drug target. Here, we report the over-expression in Escherichia coil, purification and refolding of MMP-12 catalytic domain for NMR studies. The primary sequence of expressed protein was identified by means of MALDI-TOF MS, and was confirmed by the MALDI-TOF MS data of trypsin-digested peptides. A significantly optimized protocol has been worked out to prepare 15N and/or 13C-labeled MMP-12 catalytic domain, and the yield of the purified protein is estimated to 10-12 mg from 0.5L of M9 minimal media. Finally, the 15N-1H HSQC spectrum of uniformly 15N-labeled MMP-12 catalytic domain indicates the presence of well-ordered and properly folded protein in a monomeric form.     

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.     

Mutation of a single TMVI residue, Phe(282), in the beta(2)-adrenergic receptor results in structurally distinct activated receptor conformations

We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor (alpha(1B)-AR), a residue conserved in many G protein-coupled receptors (GPCRs), is critically involved in coupling agonist binding with TM helical movement and G protein activation. Here the equivalent residue, Phe(282), in the beta(2)-AR was evaluated by mutation to glycine, asparagine, alanine, or leucine. Except for F282N, which exhibits attenuated basal and maximal isoproterenol stimulation, the Phe(282) mutants display varying degrees of constitutive activity (F282L > F282A > F282G), and as shown by the results of substituted cysteine accessibility method (SCAM) studies, induce movement of endogenous cysteine(s) into the water-accessible ligand-binding pocket. For F282A, movement is confined to Cys(285) in TMVI, whereas F282L induces movement of both Cys(285) in TMVI and Cys(327) in TMVII. Further, engineered cysteine-sensor studies indicate that F282L causes movement of TMVI, both above and below an apparent kink-inducing TMVI proline (Pro(288)), whereas that due to F282A is confined to the domain below Pro(288). A plausible interpretation of these data is that receptor activation involves rigid body movement of TMVI which, because of its Pro(288)-induced kink, acts as a pivot to transduce and amplify the agonist-induced conformational change in the upper domain, to a change in the lower domain required for productive receptor-G protein coupling.