On-chip photoactivation of heterologously expressed rhodopsin allows kinetic analysis of G-protein signaling by surface plasmon resonance spectroscopy

Surface plasmon resonance spectroscopy allows the study of protein interaction dynamics in real-time. Application of this technique to G-protein coupled receptors, the largest family of receptors involved in signal transduction, has been complicated by their low level of expression and the critical dependence of their native conformation on the hydrophobic transmembrane lipid environment. Here, we investigate and compare three different strategies to immobilize rhodopsin, a prototypical G-protein coupled receptor on a sensor chip surface using antibodies and a lectin for receptor capturing. By further probing of different experimental conditions (pH, detergent type) we identified the optimal factors to maintain rhodopsin in a functional conformation and extended this approach to recombinant rhodopsin that was heterologously expressed in COS cells. Functional operation of rhodopsin on the sensor chip surface was proven by its activation and subsequent light-stimulated G-protein coupling. The influence of these experimental parameters on the association and dissociation kinetics of G-protein receptor coupling was determined. Thereby, we found that the kinetics of G_t interaction were not changed by the strategy of immobilization or the type of detergent. Regeneration of opsin directly on a chip allowed recycling of the immobilized native and recombinant receptor. Thus, the approach provides an experimental framework for choosing the most suitable conditions for the solubilization, immobilization, and for functional tests of rhodopsin on a biosensor surface.     

Getting it together: signal transduction in G-protein coupled receptors by association of receptor domains

The mechanism of signal transduction by G-protein coupled receptors is unknown. Here, we propose that these receptors signal in a way that is qualitatively similar to that seen in the chemokine and endocrine hormone receptor families; the signal occurs when two domains of the receptor are brought together, although this is not the only requirement for signaling.     

Molecular modeling of neurokinin B and tachykinin NK₃ receptor complex

The tachykinin receptor NK? is a member of the rhodopsin family of G-protein coupled receptors. The NK? receptor has been regarded as an important drug target due to diverse physiological functions and its possible role in the pathophysiology of psychiatric disorders, including schizophrenia. The NK3 receptor is primarily activated by the tachykinin peptide hormone neurokinin B (NKB) which is the most potent natural agonist for the NK? receptor. NKB has been reported to play a vital role in the normal human reproduction pathway and in potentially life threatening diseases such as pre-eclampsia and as a neuroprotective agent in the case of neurodegenerative diseases. Agonist binding to the receptor is a critical event in initiating signaling, and therefore a characterization of the structural features of the agonists can reveal the molecular basis of receptor activation and help in rational design of novel therapeutics. In this study a molecular model for the interaction of the primary ligand NKB with its G-protein coupled receptor NK? has been developed. A three-dimensional model for the NK? receptor has been generated by homology modeling using rhodopsin as a template. A knowledge based docking of the NMR derived bioactive conformation of NKB to the receptor has been performed utilizing limited ligand binding data obtained from photoaffinity labeling and site-directed mutagenesis studies. A molecular model for the NKB-NK? receptor complex obtained sheds light on the topographical features of the binding pocket of the receptor and provides insight into the biochemical data currently available for the receptor.     

Affinity enrichment of plasma membrane for proteomics analysis

Proteomics analysis of plasma membranes from cells exposed to different extracellular environments is potentially a powerful approach for the identification of membrane-associated proteins responding to these environments. Preparation of high concentration plasma membrane fractions with low contamination from cellular organelles is essential for such studies. Here, we describe an affinity enrichment method, which combines cell surface biotinylation with affinity enrichment by immobilized streptavidin beads, for the isolation of plasma membranes. This method results in a 400-fold enrichment of plasma membrane relative to endoplasmic reticulum, a major contaminant in standard plasma membrane preparations, and dramatically reduces contamination from other cellular organelles. The biotinylation reaction did not interfere with ligand-dependent activation of receptor tyrosine kinases or G-protein coupled receptors, suggesting cell-surface signal transduction machinery remains functional. Membrane fractions prepared by this method should provide excellent starting materials for membrane proteomics analysis such as studies of dynamic trafficking and regulation of signaling molecules or identification of disease-specific membrane markers.     

TERS Detection of αVβ3 Integrins in Intact Cell Membranes

Integrins are important membrane receptors that form focal adhesions with the extracellular matrix and are transmembrane signaling proteins. We demonstrate that nanoparticles functionalized with c‐RGDfC ligands bind to intact cell membranes and selectively enhance the amino acid signals of the integrin receptor when coupled with tip‐enhanced Raman scattering (TERS) detection. Controlling the plasmonic interaction between the functionalized nanoparticle and the TERS tip provides a clear Raman signal from α_Vβ₃ integrins in the cell membrane that matches the signal of the purified integrin receptor. Random aggregation of nanoparticles on the cell does not provide the same spectral information. Chemical characterization of membrane receptors in intact cellular membranes is important for understanding membrane signaling and drug targeting. These results provide a new method to investigate the chemical interactions associated with ligand binding to membrane receptors in cells.     

Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein alpha subunits and regulator of G-protein signaling (RGS) proteins

Heterotrimeric G-proteins are molecular switches that couple serpentine receptors to intracellular effector pathways and the regulation of cell physiology. Ligand-bound receptors cause G-protein alpha subunits to bind guanosine 5'-triphosphate (GTP) and activate effector pathways. Signal termination is facilitated by the intrinsic GTPase activity of G-protein alpha subunits. Regulators of G-protein signaling (RGS) proteins accelerate the GTPase activity of the G-protein alpha subunit, and thus negatively regulate G-protein-mediated signal transduction. In vitro biochemical assays of heterotrimeric G-proteins commonly include measurements of nucleotide binding, GTPase activity, and interaction with RGS proteins. However, the conventional assays for most of these processes involve radiolabeled guanine nucleotide analogues and scintillation counting. In this article, we focus on fluorescence-based methodologies to study heterotrimeric G-protein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in high-throughput screening and drug discovery.     

Pharmacophore model for bile acids recognition by the FPR receptor

Formyl-peptide receptors (FPRs) belong to the family A of the G-protein coupled receptor superfamily and include three subtypes: FPR, FPR-like-1 and FPR-like-2. They have been involved in the control of␣many inflammatory processes promoting the recruitment and infiltration of leukocytes in regions of inflammation through the molecular recognition of chemotactic factors. A large number of structurally diverse chemotypes modulate the activity of FPRs. Newly identified antagonists include bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). The molecular recognition of these compounds at FPR receptor was computationally investigated using both ligand- and structure-based approaches. Our findings suggest that all antagonists bind at the first third of the seven helical bundles. A closer inspection of bile acid interaction reveals a number of unexploited anchor points in the binding site that may be used to aid the design of new potent and selective bile acids derivatives at FPR.     

In Silico Discovery of Antimicrobial Peptides as an Alternative to Control SARS-CoV-2

A serious pandemic has been caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The interaction between spike surface viral protein (Sgp) and the angiotensin-converting enzyme 2 (ACE2) cellular receptor is essential to understand the SARS-CoV-2 infectivity and pathogenicity. Currently, no drugs are available to treat the infection caused by this coronavirus and the use of antimicrobial peptides (AMPs) may be a promising alternative therapeutic strategy to control SARS-CoV-2. In this study, we investigated the in silico interaction of AMPs with viral structural proteins and host cell receptors. We screened the antimicrobial peptide database (APD3) and selected 15 peptides based on their physicochemical and antiviral properties. The interactions of AMPs with Sgp and ACE2 were performed by docking analysis. The results revealed that two amphibian AMPs, caerin 1.6 and caerin 1.10, had the highest affinity for Sgp proteins while interaction with the ACE2 receptor was reduced. The effective AMPs interacted particularly with Arg995 located in the S2 subunits of Sgp, which is key subunit that plays an essential role in viral fusion and entry into the host cell through ACE2. Given these computational findings, new potentially effective AMPs with antiviral properties for SARS-CoV-2 were identified, but they need experimental validation for their therapeutic effectiveness.     

Context-dependent fluorescence detection of a phosphorylated tyrosine residue by a ribonucleopeptide

Tools for selective recognition and sensing of specific phosphorylated tyrosine residues on the protein surface are essential for understanding signal transduction cascades in the cell. A stable complex of RNA and peptide, a ribonucleopeptide (RNP), provides effective approaches to tailor RNP receptors and fluorescent RNP sensors for small molecules. In vitro selection of an RNA-derived pool of RNP afforded RNP receptors specific for a phosphotyrosine residue within a defined amino-acid sequence Gly-Tyr-Ser-Arg. The RNP receptor for the specific phosphotyrosine residue was successfully converted to a fluorescent RNP sensor for sequence-specific recognition of a phosphorylated tyrosine by screening a pool of fluorescent phosphotyrosine-binding RNPs generated by a combination of the RNA subunits of phosphotyrosine-binding RNPs and various fluorophore-modified peptide subunits. The phosphotyrosine-binding RNP receptor and fluorescent RNP sensor constructed from the RNP receptor not only discriminated phosphotyrosine against tyrosine, phosphoserine, or phosphothreonine, but also showed specific recognition of amino acid residues surrounding the phosphotyrosine residue. A fluorescent RNP sensor for one of the tyrosine phosphorylation sites of p100 coactivator showed a binding affinity to the target site ~95-fold higher than the other tyrosine phosphorylation site. The fluorescent RNP sensor has an ability to function as a specific fluorescent sensor for the phosphorylated tyrosine residue within a defined amino-acid sequence in HeLa cell extracts.     

alpha(2A)-adrenergic receptor derived peptide adsorbates: a G-protein interaction study

The affinity of alpha(2A)-adrenergic receptor (alpha(2A)-AR) derived peptide adsorbates for the functional bovine brain G-protein is studied in the search for the minimum sequence recognition. Three short peptides (GPR-i2c, GPR-i3n, and GPR-i3c) are designed to mimic the second and third intracellular loops of the receptor. X-ray photoelectron spectroscopy is used to study the chemical composition of the peptides and the binding strength to the surfaces. Chemisorption of the peptides to the gold substrates is observed. Infrared spectroscopy is used to study the characteristic absorption bands of the peptides. The presence of peptides on the surfaces is verified by prominent amide I and amide II bands. The interaction between the peptides and the G-protein is studied with surface plasmon resonance. It is shown that GPR-i3n has the highest affinity for the G-protein. Equilibrium analysis of the binding shows that the G-protein keeps its native conformation when interacting with GPR-i3c, but during the interaction with GPR-i2c and GPR-i3n the conformation of G-protein is changed, leading to the formation of aggregates and/or multilayers.     

Using silico methods predicting ligands for orphan GPCRs

The G-protein coupled receptor (GPCR) superfamily is one of the most important drug target classes for the pharmaceutical industry. The completion of the human genome project has revealed that there are more than 300 potential GPCR targets of interest. The identification of their natural ligands can gain significant insights into regulatory mechanisms of cellular signaling networks and provide unprecedented opportunities for drug discovery. Much effort has been directed towards the GPCR ligand discovery study by both academic institutions and pharmaceutical industries. However, the endogenous ligands still remain unknown for about 150 GPCRs in the human genome. It is necessary to develop new strategies to predict candidate ligands for these so-called orphan receptors. Computational techniques are playing an increasingly important role in finding and validating novel ligands for orphan GPCRs (oGPCRs). In this paper, we focus on recent development in applying bioinformatics approaches for the discovery of GPCR ligands. In addition, some of the data resources for ligand identification are also provided.     

囊泡表面分子间通讯交流体系的构建: 利用受体的分子识别行为调控酶的活性

在人工双层膜囊泡表面, 构建了一个通过人工受体的分子识别行为控制酶反应活性的超分子体系. 体系以生物体细胞信号转导系统为模拟原型, 由作为受体的烷基胺、被受体识别的信号分子吡哆醛衍生物、乳酸脱氢酶、受体和酶之间的媒介物Cu^2＋以及作为体系载体的合成肽脂囊泡五个成分构成．通过UV-vis光谱法及动态光散射测定对体系进行了评价, 结果表明: 随着受体疏水参数增大, 其对信号分子的识别能力增强, 二者呈良好的线性关系; 通过信号分子与囊泡表面静电相互作用的研究表明信号分子具有选择性; 媒介物与信号分子–受体可形成化学计量比为1∶2的配合物, 其形成能力比媒介物与酶的结合能力更强．作为结论, 体系中烷基胺受体对磷酸吡哆醛信号分子的识别有效控制了处于囊泡表面的乳酸脱氢酶的活性．     

Decoding a PNA encoded peptide library by PCR: the discovery of new cell surface receptor ligands

The ability to screen and identify new ligands for cell surface receptors has been a long-standing goal as it might allow targeting of pharmaceutically relevant receptors, such as integrins or G protein coupled receptors. Here, we present a method to amplify hits from a library of PNA-tagged peptides. To this end, human cells, overexpressing either integrins or the CCR6 receptor, were treated with a 10,000 member PNA-encoded peptide library. Extraction of the PNA tags from the surface of the cells was followed by a PNA-tag to DNA translation and amplification enabling decoding of the tags via microarray hybridization. This approach to ligand discovery facilitates screening for differences in surface-receptor ligands and/or receptor expression between different cell types, and opens up a practical approach to PNA-tag amplification.     

Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together in vivo at neuromuscular junctions and in motor neurons.     

Classifying G-protein coupled receptors with bagging classification tree

G-protein coupled receptors (GPCRs) play a key role in different biological processes, such as regulation of growth, death and metabolism of cells. They are major therapeutic targets of numerous prescribed drugs. However, the ligand specificity of many receptors is unknown and there is little structural information available. Bioinformatics may offer one approach to bridge the gap between sequence data and functional knowledge of a receptor. In this paper, we use a bagging classification tree algorithm to predict the type of the receptor based on its amino acid composition. The prediction is performed for GPCR at the sub-family and sub-sub-family level. In a cross-validation test, we achieved an overall predictive accuracy of 91.1% for GPCR sub-family classification, and 82.4% for sub-sub-family classification. These results demonstrate the applicability of this relative simple method and its potential for improving prediction accuracy.     

The complexity of G-protein coupled receptor-ligand interactions

The G-protein coupled receptors (GPCRs) play fundamental roles in the human biololgy and drug discovery. GPCRs function as signalling molecules that transduce extracellular signals into cells. The signalling transduction is generally triggered by interacting with ligands, including photons, ions, small organic compounds, peptides, proteins and lipids. In this review, we focus on interactions with diffusible ligands such as hormones and neurotransmitters. We discuss three aspects of the complexity of the GPCR-ligand interactions: functional selectivity of ligands, receptor subtype selectivity of ligands and orphan GPCRs.     

Signal amplification by allosteric catalysis

In this article we unify a series of recent studies on bio- and chemosensors under a single signaling strategy: signal amplification by allosteric catalysis (SAAC). The SAAC strategy mimics biological signal transduction processes, where molecular recognition between an external signal and a protein receptor is allosterically transduced into catalytically amplified chemical information (usually second messengers). Several recent biosensing and chemosensing studies apply this nature-inspired strategy by using engineered allosteric enzymes, ribozymes, or regulatable organic catalysts. The factors pertinent to achieving high sensitivity and specificity in SAAC strategies are analyzed. The authors believe that these early studies from a variety of research groups have opened up a new venue for the development of sensing technologies where molecular recognition and catalysis can be coupled for practical purposes.     

Designer peptide surfactants stabilize diverse functional membrane proteins

Multi-spanning integral membrane proteins, including G-protein coupled receptors (GPCR), ion channels, and ion transporters, comprise a major class of drug targets. However, despite their vital importance, most molecular structures of membrane proteins remain elusive. This is largely due to lack of effective materials and methods to stabilize their functional conformation for sufficient time. Thus finding optimal surfactants and developing new approaches to study fundamental properties of unstable membrane proteins is urgently needed. In this tutorial review we summarize designer peptides with surfactant properties and their usefulness to stabilize membrane proteins. These peptide surfactants present new opportunities for the stabilization and characterization of diverse membrane proteins. Previous studies on the interaction between surfactant peptides and membrane proteins revealed strategies to design new peptides tailor-made for the stabilization of specific proteins. We review examples of solubilization, purification, long-term stabilization of membrane proteins, and the design principles of peptide sequences. We discuss future trends for exploiting spatial features, thermodynamic parameters, and self-assembling properties to create peptide surfactant structures to facilitate the characterization of diverse membrane proteins.     

Identification and characterization of mGlu3 ligands using a high throughput FLIPR assay for detection of agonists, antagonists, and allosteric modulators

When targeting G-protein coupled receptors (GPCRs) in early stage drug discovery, or for novel targets, the type of ligand most likely to produce the desired therapeutic effect may be unknown. Therefore, it can be desirable to identify potential lead compounds from multiple categories: agonists, antagonists, and allosteric modulators. In this study, we developed a triple addition calcium flux assay using FLIPR Tetra to identify multiple ligand classes for the metabotropic glutamate receptor 3 (mGlu3), using a cell line stably co-expressing the human G-protein-coupled mGlu3 receptor, a promiscuous G-protein (G(α16)), and rat Glast, a glutamate transporter. Compounds were added to the cells followed by stimulation with EC(10) and then EC(80) concentration of glutamate, the physiological agonist for mGlu receptors. This format produced a robust assay, facilitating the identification of agonists, positive allosteric modulators and antagonists/negative allosteric modulators. Follow up experiments were conducted to exclude false positives. Using this approach, we screened a library of approximately 800,000 compounds using FLIPR Tetra and identified viable leads for all three ligand classes. Further characterization revealed the selectivity of individual ligands.     

Molecular dynamics simulation involved in expounding the activation of adrenoceptors by sympathetic nervous system signaling

Structural Chemistry - G-protein–coupled receptors are integral membrane proteins involved in signal transduction pathways, making them an appealing drug targets for a wide spectrum of...