Three-dimensional structure and function study on the active region in the extracellular ligand-binding domain of human IL-6 receptor

In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hlL-6 R) was constructed by computer-guided ho-mology modeling technique using the crystal structure of the ligand-binding domain (K52-L251) of human growth hormone receptor (hGHR) as templet. Furthermore, the active binding region of the 3-D model of hlL-6R with the ligand (hlL-6) was predicted. In light of the structural characteristics of the active region, a hydrophobic pocket shielded by two hydrophilic residues (E115 and E505) of the region was identified by a combination of molecular modelling and the site-directed or double-site mutation of the twelve crucial residues in the ligand-binding domain of hIL-6R (V106-P322). We observed and analyzed the effects of these mutants on the spatial conformation of the pocket-like region of hlL-6 R. The results indicated that any site-directed mutation of the five Cys residues (four conservative Cys residues: Cyst 21, Cys132, Cys165, Cys1     

FMDV整联蛋白受体b1亚基的克隆及其配体黏附区多抗的制备

采用RT-PCR技术从牛气管组织扩增出2400 bp的b1基因, 回收纯化连入PGEM-T载体, 测序。用Expasy软件对b1基因的抗原性进行分析, 选取胞外区334~861 bp的配体结合区与6×His融合, 在大肠杆菌中大规模诱导表达, 并经Ni2+亲和柱层析纯化。通过SDS-PAGE鉴定后, 应用纯化蛋白免疫新西兰家兔, 获得效价在1:12 800以上的多抗, Western blotting鉴定表明此抗体可特异性的与表达的融合蛋白作用。     

Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Natural and synthetic inhibitors of kallikrein-related peptidases (KLKs)

Including the true tissue kallikrein KLK1, kallikrein-related peptidases (KLKs) represent a family of fifteen mammalian serine proteases. While the physiological roles of several KLKs have been at least partially elucidated, their activation and regulation remain largely unclear. This obscurity may be related to the fact that a given KLK fulfills many different tasks in diverse fetal and adult tissues, and consequently, the timescale of some of their physiological actions varies significantly. To date, a variety of endogenous inhibitors that target distinct KLKs have been identified. Among them are the attenuating Zn²⁺ ions, active site-directed proteinaceous inhibitors, such as serpins and the Kazal-type inhibitors, or the huge, unspecific compartment forming α₂-macroglobulin. Failure of these inhibitory systems can lead to certain pathophysiological conditions. One of the most prominent examples is the Netherton syndrome, which is caused by dysfunctional domains of the Kazal-type inhibitor LEKTI-1 which fail to appropriately regulate KLKs in the skin. Small synthetic inhibitory compounds and natural polypeptidic exogenous inhibitors have been widely employed to characterize the activity and substrate specificity of KLKs and to further investigate their structures and biophysical properties. Overall, this knowledge leads not only to a better understanding of the physiological tasks of KLKs, but is also a strong fundament for the synthesis of small compound drugs and engineered biomolecules for pharmaceutical approaches. In several types of cancer, KLKs have been found to be overexpressed, which makes them clinically relevant biomarkers for prognosis and monitoring. Thus, down regulation of excessive KLK activity in cancer and in skin diseases by small inhibitor compounds may represent attractive therapeutical approaches.     

Structure-based exploration of sevoflurane binding sites in cognition-related synaptotagmin-1

Synaptotagmin 1 (Syt1) is a Ca²⁺ sensor in the membrane of pre-synaptic axon terminal, which functions as an essential regulator of neurotransmitter release and hormone secretion and plays an important role in learning, memory and thinking. The sevoflurane, a general anaesthetics used widely in surgery, has been reported to modulate intracellular calcium flux and downstream neural events by targeting Syt1 C2A domain, exhibiting potential to reshape cognition. In order to explore the binding sites of sevoflurane in Syt1 C2A domain, we herein conducted a systematic computational investigation that integrated ligand pocket mapping, molecular docking calculations and molecular dynamics simulations to perform conformational sampling in the interaction space of sevoflurane with the domain. With the protocol, we were able to identify a number of ‘hotspots’ where sevoflurane can potentially bind to the domain. Subsequently, the location, geometry and physicochemical property of these putative binding sites were examined in detail using a variety of bioinformatics tools, from which three promising candidates were selected and investigated in vitro. Consequently, one was confirmed as specific binding site that can be bound tightly by sevoflurane ligand, while another was suggested to form a relatively weak, non-specific interaction with the ligand. This work would help to understand the molecular mechanism and biological implication underlying Syt1-sevoflurane recognition, and to design molecular aptamers to intervene with cognitive behaviour.     

Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF·c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSF_M) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSF_M variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSF_M structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSF_M and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSF_M residues critical for M-CSF·c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF·c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.     

Structural insights into ligand-binding pocket formation in Nurr1 by molecular dynamics simulations

Abstract

The nuclear receptor Nurr1 (NR4A2) has been identified as a potential target for the treatment of Parkinson’s disease. In contrast to most other nuclear receptors, the X-ray crystal structure of the Nurr1 ligand-binding domain (LBD) lacks any ligand-binding pocket (LBP). However, NMR spectroscopy measurements have revealed that the known Nurr1 agonist docosahexaenoic acid (DHA) binds to a region within the LBD that corresponds to the classical NR ligand-binding pocket (LBP). In order to investigate the structural dynamics of the Nurr1 LBD and to study potential LBP formation, the conformational space of the receptor was sampled using a molecular dynamics (MD) simulation. Docking of DHA into 50,000 LBD structures extracted from the simulation revealed the existence of a transient LBP that is capable to fully harbor the compound. The location of the identified pocket overlaps with the ligand-binding site suggested by NMR experiments. Structural analysis of the protein-ligand complex showed that only modest structural rearrangements within the Nurr1 LBD are required for LBP formation. These findings may support structure-based drug discovery campaigns for the development of receptor-specific agonists.     

Ecdysone and retinoid-X receptors of the blue crab, Callinectes sapidus: Cloning and their expression patterns in eyestalks and Y-organs during the molt cycle

Crustacean molting is known to be regulated largely by ecdysteroids and crustacean hyperglycemic hormone (CHH) neuropeptide family including molt-inhibiting hormone (MIH) and CHH. The surge of 20-OH ecdysone and/or ponasterone A initiates the molting process through binding to its conserved heterodimeric nuclear receptor: Ecdysone Receptor (EcR) and Ultraspiracle (USP)/Retinoid-X Receptor (RXR). To better understand the role of ecdysteroids in the molt regulation, the full-length cDNAs of the blue crab, Callinectes sapidus EcR1 and RXR1 were isolated from the Y-organs and their expression levels were determined in both Y-organs and eyestalks at various molt stages. Y-organs show the expression of four putative isoforms of CasEcRs and CasRXRs which differ in the length of the open reading frame but share the same domain structures as in typical nuclear receptors: AF1, DBD, HR, LBD, and AF2. The putative CasEcR isoforms are derived from a 27-aa insert in the HR and a 49-aa residue substitution in the LBD. In contrast, an insertion of a 5-aa and/or a 45-aa in the DBD and LBD gives rise to CasRXR isoforms. The eyestalks and Y-organs show the co-expression of CasEcRs and CasRXRs but at the different levels. In the eyestalks, the expression levels of CasRXRs are 3–5 times higher than those of CasEcRs, while in Y-organs, CasRXRs are 2.5–4 times higher than CasEcRs. A tissue-specific response to the changes in the levels of hemolymphatic ecdysteroids indicates that these tissues may have differences in the sensitivity or responsiveness to ecdysteroids. The presence of upstream open reading frame and internal ribosome entry site in 5′ UTR sequences of C. sapidus and other arthropod EcR/RXR/USP analyzed by in silico indicates a plausible, strong control(s) of the translation of these receptors.