Genetically encoded chemical probes in cells reveal the binding path of urocortin-I to CRF class B GPCR.

Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click-chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.

A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions.

Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments.

The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications.

Semisynthesis and optimization of G protein-coupled receptor mimics.

We have recently developed a soluble mimic of the corticotropin-releasing factor receptor type 1 (CRF1), a membrane-spanning G protein-coupled receptor, which allowed investigations on receptor-ligand interactions. The CRF1 mimic consists of the receptor N-terminus and three synthetic extracellular loops (ECL1-3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost-efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop-mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs.

Amyloid beta 42 peptide (Abeta42)-lowering compounds directly bind to Abeta and interfere with amyloid precursor protein (APP) transmembrane dimerization.

Following ectodomain shedding by beta-secretase, successive proteolytic cleavages within the transmembrane sequence (TMS) of the amyloid precursor protein (APP) catalyzed by gamma-secretase result in the release of amyloid-beta (Abeta) peptides of variable length. Abeta peptides with 42 amino acids appear to be the key pathogenic species in Alzheimer's disease, as they are believed to initiate neuronal degeneration. Sulindac sulfide, which is known as a potent gamma-secretase modulator (GSM), selectively reduces Abeta42 production in favor of shorter Abeta species, such as Abeta38. By studying APP-TMS dimerization we previously showed that an attenuated interaction similarly decreased Abeta42 levels and concomitantly increased Abeta38 levels. However, the precise molecular mechanism by which GSMs modulate Abeta production is still unclear. In this study, using a reporter gene-based dimerization assay, we found that APP-TMS dimers are destabilized by sulindac sulfide and related Abeta42-lowering compounds in a concentration-dependent manner. By surface plasmon resonance analysis and NMR spectroscopy, we show that sulindac sulfide and novel sulindac-derived compounds directly bind to the Abeta sequence. Strikingly, the attenuated APP-TMS interaction by GSMs correlated strongly with Abeta42-lowering activity and binding strength to the Abeta sequence. Molecular docking analyses suggest that certain GSMs bind to the GxxxG dimerization motif in the APP-TMS. We conclude that these GSMs decrease Abeta42 levels by modulating APP-TMS interactions. This effect specifically emphasizes the importance of the dimeric APP-TMS as a promising drug target in Alzheimer's disease.

Inhibition of biosynthesis of human endothelin B receptor by the cyclodepsipeptide cotransin.

The specific inhibition of the biosynthesis of target proteins is a relatively novel strategy in pharmacology and is based mainly on antisense approaches (e.g. antisense oligonucleotides or RNA interference). Recently, a novel class of substances was described acting at a later step of protein biosynthesis. The cyclic heptadepsipeptides CAM741 and cotransin were shown to inhibit selectively the biosynthesis of a small subset of secretory proteins by preventing stable insertion of the nascent chains into the Sec61 translocon complex at the endoplasmic reticulum membrane (Besemer, J., Harant, H., Wang, S., Oberhauser, B., Marquardt, K., Foster, C. A., Schreiner, E. P., de Vries, J. E., Dascher-Nadel, C., and Lindley, I. J. (2005) Nature 436, 290-293; Garrison, J. L., Kunkel, E. J., Hegde, R. S., and Taunton, J. (2005) Nature 436, 285-289). These peptides act in a signal sequence-discriminatory manner, which explains their selectivity. Here, we have analyzed the cotransin sensitivity of various G protein-coupled receptors in transfected HEK 293 cells. We show that the biosynthesis of the human endothelin B receptor (ET(B)R) is highly sensitive to cotransin, in contrast to that of the other G protein-coupled receptors analyzed. Using a novel biosynthesis assay based on fusions with the photoconvertible Kaede protein, we show that the IC(50) value of cotransin action on ET(B)R biosynthesis is 5.4 µm and that ET(B)R signaling could be completely blocked by treating cells with 30 µm cotransin. Taken together, our data add an integral membrane protein, namely the ET(B)R, to the small group of cotransin-sensitive proteins.

Light-controlled toxicity of engineered amyloid ß-peptides.

Aggregation of amyloid ß (Aß(1-42)), causing toxicity, is a critical step in Alzheimer's disease (AD). AD studies are difficult to compare because Aß(1-42) aggregation is poorly controllable under physiological conditions. To control aggregation and toxicity, we engineered light-switchable Aß(1-42) analogues that enable controllable conversion of nontoxic fibrils into toxic oligomers simply by illumination.

Mode of action of cationic antimicrobial peptides defines the tethering position and the efficacy of biocidal surfaces.

Covalent immobilization of cationic antimicrobial peptides (CAPs) at sufficient density and distance from the solid matrix has been suggested as a successful strategy for the generation of biocidal surfaces. To test the hypothesis that the mode of peptide action is decisive for the selection of an appropriate tethering position on solid surfaces, melittin (MEL), a channel-forming peptide, buforin 2 (BUF2), a peptide able to translocate bacterial membranes without permeabilization and targeting nucleic acids, and tritrpticin (TP), described to be membrane-lytic and to have intracellular targets, were C- and N-terminally immobilized on TentaGel S NH(2) resin beads as model surface. The peptide termini were modified with aminooxyacetic acid (AOA) and coupled via oxime-forming ligation. The comparison of the activities of the three peptides and their AOA-modified analogues with a KLAL model peptide which permeabilizes membranes by a so-called "carpet-like" mode provided the following results: The peptides in solution state were active against Bacillus subtilis and Escherichia coli at micromolar concentrations. MEL and TP but not BUF2-derived peptides permeabilized the inner and outer membrane of E. coli and enhanced the permeability of lipid bilayers at concentrations around their antimicrobial values (MICs). Immobilization reduced peptide activity to millimolar MICs. The activity reduction for KLAL was independent of the tethering position and comparably low, as reflected by a low ratio of MIC(tethered)/MIC(free). In contrary, the pore-forming MEL was much less active when immobilized at the N-terminus compared with the C-terminally tethered peptide. C- and N-terminal TP tethering caused an identical but much pronounced activity decrease. The tethered BUF2 peptides were inactive at the tested concentrations suggesting that the peptides could not reach the intracellular targets. In conclusion, membrane active peptides seem to be most suitable for the generation of antimicrobial surfaces, but knowledge about their mode of membrane insertion and positioning is required to identify optimal tethering positions. The relationship between the mechanism of action and position of immobilization is highly relevant for the establishment of a general approach to obtain efficient biocidal solid matrices loaded with CAPs.

Multivalent design of apoptosis-inducing bid-BH3 peptide-oligosaccharides boosts the intracellular activity at identical overall peptide concentrations.

Multivalent peptide-oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid-BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2-position of the carbohydrate units and activated for the ligation of N-terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid-BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20-fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence.

Angioprotectin: an angiotensin II-like peptide causing vasodilatory effects.

The family of angiotensin peptides has been steadily growing in recent years. Most are fragments of angiotensin II (Ang II) with different affinities to the known angiotensin receptors. Here, we describe a novel endogenous Ang II-like octapeptide in plasma from healthy humans and patients with end-stage renal failure, which acts as a stronger agonist at Mas receptors than Ang 1-7. Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an Ang II-like octapeptide, angioprotectin, with the sequence Pro-Glu-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Pro¹ and Glu² instead of Asp¹ and Arg². Pro-Glu-Val-Tyr-Ile-His-Pro-Phe in angioprotectin is most likely generated enzymatically from Ang II. Angioprotectin antagonized the contractile actions of Ang II on rat aortic rings. The physiological antagonism of vasoconstrictor actions of Ang II by angioprotectin is mediated by the Mas receptor. Angioprotectin has a stronger affinity to the Mas receptor than Ang-1-7. Plasma concentrations were ~15% of plasma Ang II concentrations in healthy volunteers and up to 50% in patients with renal failure. A commercially available Ang II antibody did not discriminate between angioprotectin and Ang II; thus, angioprotectin can contribute to Ang II concentrations measured by antibody-based assays. This novel peptide is likely to be a relevant component of the human renin-angiotensin-system.

A peptidomimetic tight junction modulator to improve regional analgesia.

The paracellular flux of solutes through tissue barriers is limited by transmembrane tight junction proteins. Within the family of tight junction proteins, claudin-1 seems to be a key protein for tightness formation and integrity. In the peripheral nervous system, the nerve fibers are surrounded with a barrier formed by the perineurium which expresses claudin-1. To enhance the access of hydrophilic pharmaceutical agents via the paracellular route, a claudin-1 specific modulator was developed. For this purpose, we designed and investigated the claudin-1 derived peptide C1C2. It transiently increased the paracellular permeability for ions and high and low molecular weight compounds through a cellular barrier model. Structural studies revealed a ß-sheet potential for the functionality of the peptide. Perineurial injection of C1C2 in rats facilitated the effect of hydrophilic antinociceptive agents and raised mechanical nociceptive thresholds. The mechanism is related to the internalization of C1C2 and to a vesicle-like distribution within the cells. The peptide mainly colocalized with intracellular claudin-1. C1C2 decreased membrane-localized claudin-1 of cells in culture and in vivo in the perineurium of rats after perineurial injection. In conclusion, a novel tool was developed to improve the delivery of pharmaceutical agents through the perineurial barrier by transient modulation of claudin-1.

Intramolecular bridges formed by photoswitchable click amino acids.

Photoswitchable click amino acids (PSCaa) are amino acids bearing a side chain consisting of a photoswitchable unit elongated with a functional group that allows for a specific click reaction, such as an alkene that can react with the thiol group of a cysteine residue. An intramolecular click reaction results in the formation of a photoswitchable bridge, which can be used for controlling conformational domains in peptides and proteins. The ability to control conformations as well as the efficiency of the intramolecular bridging depends on the length of the PSCaa side chain and the distance to the cysteine residue to be clicked with. On comparing i,i+4 and i,i+7 spacings of PSCaa and cysteine in a model peptide without a preferred conformation, it was seen that the thiol-ene click reaction takes place efficiently in both cases. Upon induction of an α-helical structure by the addition of trifluoroethanol, the thiol click reaction occurs preferentially with the i,i+4 spacing. Even in the presence of glutathione as an additional thiol the click reaction of the PSCaa occurs intramolecularly with the cysteine rather than with the glutathione, indicating that the click reaction may be used even under reducing conditions occurring in living cells.

Directed evolution of sortase A mutants with altered substrate selectivity profiles.

The ligation of two polypeptides in a chemoselective manner by the bacterial transpeptidase sortase A has become a versatile tool for protein engineering approaches. When sortase-mediated ligation is used for protein semisynthesis, up to four mutations resulting from the strict requirement of the LPxTG sorting motif are introduced into the target protein. Here we report the directed evolution of a mutant sortase A that possesses broad substrate selectivity. A phage-display screen of a mutant sortase library that was randomized in the substrate recognition loop was used to isolate this mutant. The altered substrate selectivity represents a gain-of-function that was exploited for the traceless semisynthesis of histone H3. Our report is a decisive step toward a platform of engineered sortases with distinct ligation properties that will conceivably allow for more versatile assemblies of modified proteins in biotechnological approaches.

Photoswitchable click amino acids: light control of conformation and bioactivity.

Click the switch: By using a photoswitchable click amino acid (PSCaa) a light-induced intramolecular thiol-ene click reaction with a neighboring cysteine under very mild conditions results in an azobenzene bridge. By expanding the genetic code for PSCaa the specific incorporation of photoswitch units into proteins in living cells can result in an exciting approach for studying light-controllable activity, in vivo.

The signal flow and motor response controling chemotaxis of sea urchin sperm.

The signalling pathway and the behavioural strategy underlying chemotaxis of sperm are poorly understood. We have studied the cellular events and motor responses that mediate chemotaxis of sperm from the sea urchin Arbacia punctulata. Here we show that resact, a chemoattractant peptide, initiates a rapid and transient rise in the concentration of cyclic GMP, followed by a transient influx of Ca2+. The binding of a single resact molecule elicits a Ca2+ response, and 50-100 bound molecules saturate the response. The ability to register single molecules is reminiscent of the single-photon sensitivity of rod photoreceptors. Both resact and cyclic nucleotides cause a turn or brief tumbling in the swimming path of sperm. We conclude that a cGMP-mediated increase in the Ca2+ concentration induces the primary motor response of sperm to the chemoattractant.

Role of amyloid-beta glycine 33 in oligomerization, toxicity, and neuronal plasticity.

The aggregation of the amyloid-beta (Abeta) peptide plays a pivotal role in the pathogenesis of Alzheimer's disease, as soluble oligomers are intimately linked to neuronal toxicity and inhibition of hippocampal long-term potentiation (LTP). In the C-terminal region of Abeta there are three consecutive GxxxG dimerization motifs, which we could previously demonstrate to play a critical role in the generation of Abeta. Here, we show that glycine 33 (G33) of the central GxxxG interaction motif within the hydrophobic Abeta sequence is important for the aggregation dynamics of the peptide. Abeta peptides with alanine or isoleucine substitutions of G33 displayed an increased propensity to form higher oligomers, which we could attribute to conformational changes. Importantly, the oligomers of G33 variants were much less toxic than Abeta(42) wild type (WT), in vitro and in vivo. Also, whereas Abeta(42) WT is known to inhibit LTP, Abeta(42) G33 variants had lost the potential to inhibit LTP. Our findings reveal that conformational changes induced by G33 substitutions unlink toxicity and oligomerization of Abeta on the molecular level and suggest that G33 is the key amino acid in the toxic activity of Abeta. Thus, a specific toxic conformation of Abeta exists, which represents a promising target for therapeutic interventions.

Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin A receptor.

G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems.

Design, synthesis, structure and binding properties of PDZ binding, cyclic beta-finger peptides.

Protein interaction domains (PIDs) play a critical role in signal transduction. One PID of great interest is the PDZ domain, a 100 amino-acid-residue domain. Most PDZ domains recognize short, C-terminal peptide motives. In the heterodimer of the nNOS-PDZ domain and the alpha-syntrophin-PDZ domain, however, one PDZ domain forms a beta-finger that binds to the other PDZ domain. We show here that cyclic peptides derived from the beta-finger of the nNOS-PDZ domain can bind the syntrophin-PDZ domain in the same manner as the whole domain. The structure of three "finger-peptides" of different size has been determined and the binding investigated using calorimetry and NMR-titration experiments.

Insight into the role of HSPG in the cellular uptake of apolipoprotein E-derived peptide micelles and liposomes.

Liposomes and micellar carriers equipped with targeting and cellular uptake mediating peptides have attracted attention for numerous applications. The optimization of the carrier requires an understanding of how their properties influence target cell recognition and uptake. We developed a dipalmitoylated apolipoprotein E-derived peptide, named P2A2 as promising vector to mediate cellular uptake of potential micellar and liposomal carriers. Confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) were used to get insight into the internalization mediated by carboxyfluoresceine-labeled P2fA2 and the all-D amino acid analogue P2fa2 into brain capillary endothelial cells. Both peptide micelles and liposomes entered cells via endocytosis. Cell surface heparan sulfate proteoglycans (HSPGs) were involved in the internalization process of peptide-bearing liposomes characterized by a diameter of 100 nm, a low surface density of 100 peptide molecules per vesicle and a helical conformation of the vector. In contrast, peptide micelles characterized by a diameter of about 10 nm, a high peptide density caused by 19 associated molecules and a high conformational flexibility of the vector sequence did not address HSPG. Unspecific interactions between the carriers and membrane constituents predominate the two uptake processes but stereospecific components seem to be involved. Both routes differ with respect to transport efficiency. The results provide a prospective basis to optimize liposomes and micelles as drug delivery systems.