Biosynthesis and spectroscopic characterization of 2-TM fragments encompassing the sequence of a human GPCR, the Y4 receptor

This paper presents a divide‐and‐conquer approach towards obtaining solution structures of G protein‐coupled receptors. The human Y4 receptor was dissected into two to three transmembrane helix fragments, which were individually studied by solution NMR. We systematically compared various biosynthetic routes for the expression of the fragments in Escherichia coli and discuss purification strategies. In particular, we have compared the production of transmembrane (TM) fragments as inclusion bodies by using the ΔTrp leader sequence, with membrane‐directed expression by using Mistic as the fusion partner, and developed methods for enzymatic cleavage. In addition, direct expression of two‐TM fragments into inclusion bodies is a successful route in some cases. With the exception of TM13, we could produce all fragments in isotope‐labeled form in quantities sufficient for NMR studies. Almost complete backbone resonance assignment was obtained for the first two helices, as well as for helices 5 and 7, and a high degree was obtained for TM6, while conformational exchange processes resulted in the disappearance of many signals from TM4. In addition, complete assignments were obtained for all residues of the N‐terminal domain, as well as the extracellular and cytosolic loops (with the exception of an undecapeptide segment in the second extracellular loop, EC2) and for the complete cytosolic C‐terminal tail. In total, backbone resonances of 78 % of all residues were assigned for the Y4 receptor. Predictions of secondary structure based on backbone chemical shifts indicate that most residues from the TM regions adopt helical conformations, with exception of those around polar residues or prolines. However, the domain boundaries differ slightly from those predicted for homology models. We suggest that the obtained chemical shifts might be useful in assigning the full‐length receptor.     

Impact of helix irregularities on sequence alignment and homology modeling of G protein-coupled receptors

Comparison of the crystal structures of G protein-coupled receptors (GPCRs) revealed backbone irregularities in the majority of the transmembrane (TM) helices. Among these, wide (π bulge) and tight (3(10)) helical turns on TM2 and TM5 deserve special attention because of their proximity to the ligand binding site. These irregularities are related to residue insertion or deletion (reflected by inclusion of gaps in sequence alignments) accumulated during the evolution of these two helices. These findings have direct implications for the sequence alignments, phylogeny reconstruction, and homology modeling of class A GPCRs.     

Structural models of the photointermediates in the rhodopsin photocascade, lumirhodopsin, metarhodopsin I, and metarhodopsin II

Model building of the two photointermediates, lumirhodopsin and metarhodopsin I, and the activated form of rhodopsin, metarhodopsin II, is described. An outward swing of the C-terminal portion of transmembrane segment 3, pivoting on Cys110 at the N-terminal end of transmembrane segment 3, led to structural models of lumirhodopsin and metarhodopsin I. The conformation of the chromophore in the lumirhodopsin and metarhodopsin I models is controlled by the motion of transmembrane segment 3 and agreed closely with the hydrogen-bonding states of the protonated Schiff base in lumirhodopsin and metarhodopsin I as deduced from their FTIR and resonance Raman spectra and with the negative and positive CD bands of lumirhodopsin and metarhodopsin I, respectively. The structure of metarhodopsin II was constructed by an outward swing of transmembrane segment 3 and the rigid-body motion of transmembrane segment 6. The arrangement of the entire transmembrane segment of the metarhodopsin II model closely agreed with the electron paramagnetic resonance spectra of spin-labeled rhodopsin mutants and provided a structural basis for the protonation of Glu134, which is a key process in transducin activation.     

Interactions of HIV‐1 Antibodies 2F5 and 4E10 with a gp41 Epitope Prebound to Host and Viral Membrane Model Systems

Point of recognition : Surface plasmon resonance was used to study the role of the viral membrane in HIV immunogen recognition by the monoclonal antibodies 2F5 and 4E10. The different behaviour of the antibodies towards membranes and immunogens embedded within membranes, provide insight into the emerging membrane‐based or membrane‐conformation strategies of immunogen design.

     

Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor

The Eph-ephrin system, including the EphA2 receptor and the ephrinA1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist that is able to inhibit EphA2 receptor activation; it is therefore potentially useful as a novel EphA2 receptor-targeting agent. Herein we explore the structure-activity relationships of a focused set of lithocholic acid derivatives based on molecular modeling investigations and displacement binding assays. Our exploration shows that while the 3-α-hydroxy group of lithocholic acid has a negligible role in recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrinA1 to EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits the EphA2-ephrinA1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand binding domain of EphA2, with a steady-state dissociation constant (K(D) ) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 as well as cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrinA1 ligand. These findings suggest that cholanic acid can be used as a template structure for the design of effective EphA2 antagonists, and may have potential impact in the elucidation of the role played by this receptor in pathological conditions.     

Prediction of the 3D structure of FMRF-amide neuropeptides bound to the mouse MrgC11 GPCR and experimental validation

We report the 3D structure predicted for the mouse MrgC11 (mMrgC11) receptor by using the MembStruk computational protocol, and the predicted binding site for the F-M-R-F-NH(2) neuropeptide together with four singly chirally modified ligands. We predicted that the R-F-NH(2) part of the tetrapeptide sticks down into the protein between the transmembrane (TM) domains 3, 4, 5, and 6. The Phe (F-NH(2)) interacted favorably with Tyr110 (TM3), while the Arg makes salt bridges to Asp161 (TM4) and Asp179 (TM5). We predicted that the Met extends from the binding site, but the terminal Phe residue sticks back into an aromatic/hydrophobic site flanked by Tyr237, Leu238, Leu240, and Tyr256 (TM6), and Trp162 (TM4). We carried out subsequent mutagenesis experiments followed by intracellular calcium-release assays that demonstrated the dramatic decrease in activity for the Tyr110Ala, Asp161Ala, and Asp179Ala substitutions, which was predicted by our model. These experiments provide strong evidence that our predicted G protein-coupled receptor (GPCR) structure is sufficiently accurate to identify binding sites for selective ligands. Similar studies were made with the mMrgA1 receptor, which did not bind the R-F-NH(2) dipeptide; we explain this to be due to the increased hydrophobic character of the binding pocket in mMrgA1.     

Synthesis and evaluation of a fluorescent non-peptidic cholecystokinin-B/gastrin receptor specific antagonist for cancer cell imaging

Fluorescent labeling has enabled a better understanding of the relationships between receptor location, function, and life cycle. Each of these perspectives contributes new insights into drug action, particularly for G protein‐coupled receptors (GPCRs). The aim of this study was to develop a fluorescein derivative, FLUO‐QUIN—a novel antagonist of the cholecystokinin‐B/gastrin receptor. A radioligand‐binding experiment revealed an IC₅₀ of 4.79 nm, and the antagonist inhibited gastric acid secretion in an isolated lumen‐perfused mouse stomach assay (up to 51 % at 100 nm) . The fluorescence properties altered upon binding to the receptor, and the fluorophore was quenched to a greater extent when free than in the bound form. FLUO‐QUIN specifically bound to human pancreatic carcinoma cells, MiaPaca‐2, which are known to express the receptor, as evidenced by rapid clustering followed by time‐dependent receptor internalization. This proves the stability of FLUO‐QUIN and its ability to penetrate vesicular membranes and reach various cell targets. Hence it might be used as an agent for the detection of CCK‐B‐receptor‐positive tumors by fluorescence imaging.     

Evaluation of a fluorescent derivative of AMD3100 and its interaction with the CXCR4 chemokine receptor

AMD3100 is a potent and selective antagonist of the CXCR4 receptor; it has been shown to block the route of entry of HIV into host T-cells. This compound and its analogues have since been found to act as haematopoietic stem cell mobilisation agents and, more recently, as anti-cancer agents. Here, we have examined a fluorescent derivative of AMD3100, L(1), which offered the potential to assess the behaviour of AMD3100 at the cell surface by using optical imaging modalities. The binuclear Zn(II) , Cu(II) and Ni(II) complexes of L(1) have also been investigated as these metals have been previously shown to enhance the binding properties of AMD3100. Furthermore, Zn(II) and Cu(II) are known to enhance and quench, respectively, the fluorescence of similar anthracenyl-based ligands. Whilst L(1) demonstrates an ability to inhibit the binding of the anti-CXCR4 monoclonal antibody 12G5 (IC(50) =0.25-0.9 μM), the incorporation of an anthracenyl moiety resulted in a significantly reduced affinity for CXCR4 compared to AMD3100 (IC(50) =10 nM). We observed no significant increase in fluorescence intensity following incubation with murine pre-B cells overexpressing CXCR4 compared to a control cell line. This limits the usefulness of L(1) as a fluorescent imaging probe. Interestingly, the Zn(II) complex, which carries an overall +4 charge, revealed marginally higher specificity and reduced toxicity in vitro compared to the free ligand, albeit with reduced affinity for CXCR4 (IC(50) =1.8-5 μM). We suggest that the incorporation of an anthracenyl group contributes to the lipophilic character of the free ligand, thereby resulting in transport across the plasma membrane. This effect is seemingly diminished when the ligand is complexed to charged metal ions.     

Surface-plasmon-resonance-based chemical proteomics: efficient specific extraction and semiquantitative identification of cyclic nucleotide-binding proteins from cellular lysates by using a combination of surface plasmon resonance, sequential elution and liquid chromatography-tandem mass spectrometry

Chemical proteomics is a powerful methodology for identifying the cellular targets of small molecules, however, it is biased towards abundant proteins. Therefore, quantitative strategies are needed to distinguish between specific and nonspecific interactions. Here, we explore the potential of the combination of surface plasmon resonance (SPR) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) as an alternative approach in chemical proteomics. We coupled cGMP molecules to the SPR chip, and monitored the binding and dissociation of proteins from a human lysate by using sequential elution steps and SPR. The eluted proteins were subsequently identified by LC-MS/MS. Our approach enabled the efficient and selective extraction of low-abundant cyclic-nucleotide-binding proteins such as cGMP-dependent protein kinase, and a quantitative assessment of the less- and nonspecific competitive binding proteins. The data show that SPR-based chemical proteomics is a promising alternative for the efficient specific extraction and quantitative identification of small-molecule-binding proteins from complex mixtures.     

Pervaporative enrichment of 2,3‐butanediol from its mixture with 1‐butanol using a polydimethylsiloxane and ZSM‐5 mixed matrix membrane: Effects of ethanol as a by‐product

A ZSM‐5 filled polydimethylsiloxane membrane with 44.4 wt.% zeolite loading was used in the pervaporative removal of 1‐butanol from its mixtures with 1‐butanol. A small quantity of ethanol was added to the feed as a by‐product to test the response of the membrane. It was found that the permeation behaviour of other feed components was changed and membrane selectivity decreased. This change was attributed to the frequently‐observed inter‐component coupled transport in multi‐component feed systems. The impact of ethanol on recovery of 2,3‐butanediol was evaluated using a simulated continuous operation, which enriched 2,3‐butanediol to 99.5 wt.% from a feed containing 5 wt.% 2,3‐butanediol and less than 1.0 wt.% ethanol. It was observed that membrane selectivity improves as ethanol concentration decreases in the stream due to its preferential removal. The final recovery of 2,3‐butanediol was not significantly reduced as the concentration of ethanol was below 1.0 wt.%. © 2011 Canadian Society for Chemical Engineering