Short Oligourea Foldamers as N- or C-Caps for Promoting α-Helix Formation in Water.

While foldamers have been extensively studied as protein mimics and especially as α-helix mimics, their use as capping motif to enhance α-helix propensity remains comparatively much limited. In this study, we leverage the structural similarities between urea-based helical foldamers and α-helix to investigate the efficacy of oligoureas as N- or C-caps for reinforcing α-helical structures in water. Short oligoureas, comprising 3 to 4 residues, were strategically introduced at the N- or C-terminus of two peptide sequences (S-peptide and an Ala-rich model sequence). The impact of these foldamer insertions on peptide conformation was examined using electronic circular dichroism (ECD) and solution NMR. This research identifies specific foldamer sequences capable of promoting a-helicity when incorporated at either terminus of the peptides. Not only does this work broaden the application scope of foldamers, but it also provides valuable insights into novel strategies for modulating peptide conformation in aqueous environments. The findings presented in this study may have implications for peptide design and the development of bioactive foldamer-based peptide mimics.

1H, 13C, 15N NMR resonance assignments and secondary structure determination of the extra-cellular domain from the human proapoptotic TRAIL-R2 death receptor 5 (DR5-ECD).

Death receptors (DR) selectively drive cancer cells to apoptosis upon binding to the Tumor necrosis factor-a-Related Apoptosis-Inducing Ligand (TRAIL). Complex formation induces the oligomerization of the death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) and transduces the apoptogenic signal to their respective death domains, leading to Death Inducing Signaling Complex (DISC) formation, caspase activation and ultimately cell death. Several crystal structures of the ExtraCellular Domain from Death Receptor 5 (DR5-ECD) have been reported in complex with the TRAIL ligand or anti-DR5 antibodies, but none for the isolated protein. In order to fill this gap and to perform binding experiments with TRAIL peptidomimetics, we have produced isotopically labelled DR5-ECD and started a conformational analysis by using high-field 3D NMR spectroscopy. Herein, we present the first resonance assignment of a TRAIL receptor in solution and the determination of its secondary structure from NMR chemical shifts.

Assignment of 1H, 13C and 15N resonances and secondary structure of the Rgd1-RhoGAP domain.

The protein Rgd1 is involved in the regulation of cytoskeleton formation and in signalling pathways that control cell polarity and growth in Saccharomyces cerevisiae. Rgd1p is composed of a F-BAR domain required for membrane binding and a RhoGAP domain responsible for activating Rho3p and Rho4p, two GTPases respectively involved in bud growth and cytokinesis. Rgd1p is recruited to the membrane through interactions with phosphoinositide lipids, which bind the two isolated domains and stimulate the RhoGAP activity on Rho4p. As previously shown by crystallography, the membrane-binding F-BAR domain contains a conserved inositol phosphate binding site, which explains the preferential binding of phosphoinositides. In contrast, RhoGAP domains are not expected to bind lipids. In order to unravel this puzzling feature, we solved the three-dimensional structure of the isolated protein and found a cryptic phosphoinositide binding site involving non conserved residues (Martinez et al. 2017). The assignment of the resonances and secondary structure of Rgd1-RhoGAP (aa 450-666) is presented here.

Structural evidence of a phosphoinositide-binding site in the Rgd1-RhoGAP domain.

Phosphoinositide lipids recruit proteins to the plasma membrane involved in the regulation of cytoskeleton organization and in signalling pathways that control cell polarity and growth. Among those, Rgd1p is a yeast GTPase-activating protein (GAP) specific for Rho3p and Rho4p GTPases, which control actin polymerization and stress signalling pathways. Phosphoinositides not only bind Rgd1p, but also stimulate its GAP activity on the membrane-anchored form of Rho4p. Both F-BAR (F-BAR FCH, and BAR) and RhoGAP domains of Rgd1p are involved in lipid interactions. In the Rgd1p-F-BAR domain, a phosphoinositide-binding site has been recently characterized. We report here the X-ray structure of the Rgd1p-RhoGAP domain, identify by NMR spectroscopy and confirm by docking simulations, a new but cryptic phosphoinositide-binding site, comprising contiguous A1, A1' and B helices. The addition of helix A1', unusual among RhoGAP domains, seems to be crucial for lipid interactions. Such a site was totally unexpected inside a RhoGAP domain, as it was not predicted from either the protein sequence or its three-dimensional structure. Phosphoinositide-binding sites in RhoGAP domains have been reported to correspond to polybasic regions, which are located at the unstructured flexible termini of proteins. Solid-state NMR spectroscopy experiments confirm the membrane interaction of the Rgd1p-RhoGAP domain upon the addition of PtdIns(4,5)P2 and indicate a slight membrane destabilization in the presence of the two partners.

Cell-Free Expression for the Study of Hydrophobic Proteins: The Example of Yeast ATP-Synthase Subunits.

Small hydrophobic membrane proteins or proteins with hydrophobic domains are often difficult to produce in bacteria. The cell-free expression system was found to be a very good alternative for the expression of small hydrophobic subunits of the yeast ATP-synthase, such as subunits e, g, k, i, f and the membrane domain of subunit 4, proteins that are suspected to play a role in the stability of ATP-synthase dimers. All of these proteins could be produced in milligrams amounts using the cell-free "precipitate mode" and were successfully solubilized in the presence of lysolipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-1'-rac-glycerol. Purified proteins were also found suitable for structural investigations. An example is given with the NMR backbone assignment of the isotopically labeled subunit g. Protocols are also described for raising specific polyclonal antibodies against overexpressed cell-free proteins.

α-Peptide-Oligourea Chimeras: Stabilization of Short α-Helices by Non-Peptide Helical Foldamers.

Short α-peptides with less than 10 residues generally display a low propensity to nucleate stable helical conformations. While various strategies to stabilize peptide helices have been previously reported, the ability of non-peptide helical foldamers to stabilize α-helices when fused to short α-peptide segments has not been investigated. Towards this end, structural investigations into a series of chimeric oligomers obtained by joining aliphatic oligoureas to the C- or N-termini of α-peptides are described. All chimeras were found to be fully helical, with as few as 2 (or 3) urea units sufficient to propagate an α-helical conformation in the fused peptide segment. The remarkable compatibility of α-peptides with oligoureas described here, along with the simplicity of the approach, highlights the potential of interfacing natural and non-peptide backbones as a means to further control the behavior of α-peptides.

Thioether analogues of disulfide-bridged cyclic peptides targeting death receptor 5: conformational analysis, dimerisation and consequences for receptor activation.

Cyclic peptides containing redox-stable thioether bridges might provide a useful alternative to disulfide-bridged bioactive peptides. We report the effect of replacing the disulfide bridge with a lanthionine linkage in a 16-mer cyclic peptide that binds to death receptor 5 (DR5, TRAIL-R2). Upon covalent oligomerisation, the disulfide-bridged peptide has previously shown similar behaviour to that of TNF-related apoptosis inducing ligand (TRAIL), by selectively triggering the DR5 cell death pathway. The structural and biological properties of the DR5-binding peptide and its desulfurised analogue were compared. Surface plasmon resonance (SPR) data suggest that these peptides bind DR5 with comparable affinities. The same holds true for dimeric versions of these peptides: the thioether is able to induce DR5-mediated apoptosis of BJAB lymphoma and tumorigenic BJELR cells, albeit to a slightly lower extent compared to its disulfide homologue. NMR analysis revealed subtle variation in the conformations of the two peptides and suggests that the thioether peptide is slightly less folded than its disulfide homologue. These observations could account for the different capability of the two dimers to cluster DR5 receptors on the cell surface and to trigger apoptosis. Nevertheless, our results suggest that the thioether peptide is a potential candidate for evaluation in animal models.

Membrane interacting peptides: from killers to helpers.

Membrane interacting peptides are reviewed in terms of structure and mode of action on lipid membranes. Helical, ß-stranded, peptides containing both helices and strands, cyclic, lipopeptides and short linear peptides are seen to considerably modulate membrane function. Among peptides that lead to membrane alteration or permeation, antimicrobial peptides play an important role and some of them may be foreseen as potential new antibiotics. Alternatively, peptides that do not destroy the membrane are also very important in modulating the structure and dynamics of the lipid bilayer and play important roles in membrane protein functions. Peptide lipid complexes are shown to be very variable in structure and dynamics: "carpet", "barrel stave", toroid and disordered pores, electrostatic wedge and molecular electroporation models are discussed. Their assembly is reviewed in terms of electric, amphipathic and dynamic properties of both lipids and peptides.

Evidence for specific interaction between the RhoGAP domain from the yeast Rgd1 protein and phosphoinositides.

The Rho GTPase activating protein Rgd1 increases the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p is a member of the F-BAR family conserved in eukaryotes; indeed, in addition to the C-terminal RhoGAP domain Rgd1p possesses an F-BAR domain at its N-terminus. Phosphoinositides discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity of Rgd1p on Rho4p. Determining specific interactions and resolving the structure of Rgd1p should provide insight into the functioning of this family of protein. We report the preparation of highly pure and functional RhoGAP domain of Rgd1 RhoGAP domain using a high yield expression procedure. By gel filtration and circular dichroïsm we provide the first evidences for a specific interaction between a RhoGAP domain (the RhoGAP domain of Rgd1p) and phosphoinositides.

Structure-activity relationship of human liver-expressed antimicrobial peptide 2.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a 40-residue cationic peptide originally purified from human blood ultrafiltrate. The native peptide contains two disulfide bonds and is unique regarding its primary structure. Its biological role is not known but a previous study showed that chemically synthesized LEAP-2 exhibited in vitro antimicrobial activities against several Gram-positive bacteria. In order to determine its antimicrobial mode of action, we expressed human recombinant LEAP-2 in Escherichia coli. Circular dichroism spectroscopy and nuclear magnetic resonance analyses showed that the structure of the recombinant peptide was identical to that of the chemically synthesized and oxidized LEAP-2, with two disulfide bonds between Cys residues in relative 1-3 and 2-4 positions. Minimal inhibitory concentration (MIC) of the recombinant human LEAP-2 was determined by a conventional broth dilution assay. It was found to be bactericidal against Bacillus megaterium at a 200microM concentration. Interestingly, the linear LEAP-2 had a greater antimicrobial activity with a MIC value of 12.5microM, which was comparable to that of magainin2. SYTOX Green uptake was used to assess bacterial membrane integrity. Linear LEAP-2 and magainin2 permeabilized B. megaterium membranes with the same efficiency, whereas oxidized LEAP-2 did not induce stain uptake. Binding of the peptides to plasmid DNA was evaluated by gel retardation assays. The DNA-binding efficacy of linear LEAP-2 was three times higher than that of the peptide-containing disulfide bridges. Altogether, these results show that the secondary structure of human LEAP-2 has a profound impact on its antibacterial activity.

Specific interactions of clausin, a new lantibiotic, with lipid precursors of the bacterial cell wall.

We investigated the specificity of interaction of a new type A lantibiotic, clausin, isolated from Bacillus clausii, with lipid intermediates of bacterial envelope biosynthesis pathways. Isothermal calorimetry and steady-state fluorescence anisotropy (with dansylated derivatives) identified peptidoglycan lipids I and II, embedded in dodecylphosphocholine micelles, as potential targets. Complex formation with dissociation constants of approximately 0.3 muM and stoichiometry of approximately 2:1 peptides/lipid intermediate was observed. The interaction is enthalpy-driven. For the first time, to our knowledge, we evidenced the interaction between a lantibiotic and C(55)-PP-GlcNAc, a lipid intermediate in the biosynthesis of other bacterial cell wall polymers, including teichoic acids. The pyrophosphate moiety of these lipid intermediates was crucial for the interaction because a strong binding with undecaprenyl pyrophosphate, accounting for 80% of the free energy of binding, was observed. No binding occurred with the undecaprenyl phosphate derivative. The pentapeptide and the N-acetylated sugar moieties strengthened the interaction, but their contributions were weaker than that of the pyrophosphate group. The lantibiotic decreased the mobility of the pentapeptide. Clausin did not interact with the water-soluble UDP-MurNAc- and pyrophosphoryl-MurNAc-pentapeptides, pointing out the importance of the hydrocarbon chain of the lipid target.

Aggregation of cateslytin beta-sheets on negatively charged lipids promotes rigid membrane domains. A new mode of action for antimicrobial peptides?

Cateslytin, a positively charged (5+) arginine-rich antimicrobial peptide (bCgA, RSMRLSFRARGYGFR), was chemically synthesized and studied against membranes that mimic bacterial or mammalian systems. Circular dichroism, polarized attenuated total reflection infrared spectroscopy, (1)H high-resolution MAS NMR, and (2)H and (31)P solid state NMR were used to follow the interaction from peptide and membrane points of view. Cateslytin, which is unstructured in solution, is converted into antiparallel beta-sheets that aggregate mainly flat at the surface of negatively charged bacterial mimetic membranes. Arginine residues are involved in the binding to negatively charged lipids. Following the interaction of the cateslytin peptide, rigid and thicker membrane domains enriched in negatively charged lipids are found. Much less interaction is detected with neutral mammalian model membranes, as reflected by only minor percentages of beta-sheets or helices in the peptide secondary structure. No membrane destruction was detected for both bacterial and mammalian model membranes. A molecular model is proposed in which zones of different rigidity and thickness bring about phase boundary defects that ultimately lead to permeability induction and peptide crossing through bacterial membranes.

S1 ribosomal protein functions in translation initiation and ribonuclease RegB activation are mediated by similar RNA-protein interactions: an NMR and SAXS analysis.

The ribosomal protein S1, in Escherichia coli, is necessary for the recognition by the ribosome of the translation initiation codon of most messenger RNAs. It also participates in other functions. In particular, it stimulates the T4 endoribonuclease RegB, which inactivates some of the phage mRNAs, when their translation is no longer required, by cleaving them in the middle of their Shine-Dalgarno sequence. In each function, S1 seems to target very different RNAs, which led to the hypothesis that it possesses different RNA-binding sites. We previously demonstrated that the ability of S1 to activate RegB is carried by a fragment of the protein formed of three consecutive domains (domains D3, D4, and D5). The same fragment plays a central role in all other functions. We analyzed its structural organization and its interactions with three RNAs: two RegB substrates and a translation initiation region. We show that these three RNAs bind the same area of the protein through a set of systematic (common to the three RNAs) and specific (RNA-dependent) interactions. We also show that, in the absence of RNA, the D4 and D5 domains are associated, whereas the D3 and D4 domains are in equilibrium between open (noninteracting) and closed (weakly interacting) forms and that RNA binding induces a structural reorganization of the fragment. All of these results suggest that the ability of S1 to recognize different RNAs results from a high adaptability of both its structure and its binding surface.

Variability in secondary structure of the antimicrobial peptide Cateslytin in powder, solution, DPC micelles and at the air-water interface.

Cateslytin (bCGA (344)RSMRLSFRARGYGFR(358)), a five positively charged 15 amino-acid residues arginine-rich antimicrobial peptide, was synthesized using a very efficient procedure leading to high yields and to a 99% purity as determined by HPLC and mass spectrometry. Circular dichroism, polarized attenuated total reflectance fourier transformed infrared, polarization modulation infrared reflection Absorption spectroscopies and proton two-dimensional NMR revealed the flexibility of such a peptide. Whereas being mostly disordered as a dry powder or in water solution, the peptide acquires a alpha-helical character in the "membrane mimicking" solvent trifuoroethanol. In zwitterionic micelles of dodecylphophatidylcholine the helical character is retained but to a lesser extent, the peptide returning mainly to its disordered state. A beta-sheet contribution of almost 100% is detected at the air-water interface. Such conformational plasticity is discussed regarding the antimicrobial action of Cateslytin.

RNA recognition and cleavage by sequence-specific endoribonucleases.

Inactivation of RNA molecules by sequence-specific endoribonucleolytic cleavage is a subtle mechanism by which cells regulate gene expression. Sequence-specific endoribonucleases can recognize and cleave particular phosphodiester bonds confined within hundreds/thousands of chemically similar bonds. Here, we present a comparative analysis of the mechanisms used by endoribonucleases to select and cleave their target RNA molecules. This analysis is based on the very recent molecular details obtained from the structural and/or biochemical studies of nine sequence-specific ribonucleases that target messenger, ribosomal, and transfer RNA molecules. This analysis shows that despite the absence of sequence homologies and the wide diversity of biological sources (prokaryotes, archaea and eukaryotes), the sequence-specific ribonucleases studied here adopt limited structural folds, catalyze their cleavage reactions using a common chemistry and involve a very limited set of amino acids for both RNA binding and processing.

Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome.

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.

1H, 13C and 15N resonance assignment of phage T4 endoribonuclease RegB.

RegB is involved in the control of the phage T4 life cycle. It inactivates the phage early mRNAs when their translation is no more required. We determined its structure and identified residues involved in substrate binding. For this, all backbone and 90% of side-chain resonance frequencies were assigned.

First structural investigation of the restriction ribonuclease RegB: NMR spectroscopic conditions, 13C/15N double-isotopic labelling and two-dimensional heteronuclear spectra.

The bacteriophage T4 genome-encoded ribonuclease RegB is the unique well-defined restriction endoribonuclease. This protein cleaves with an almost absolute specificity its RNA substrate in the middle of the GGAG tetranucleotide mainly found in the Shine-Dalgarno sequence (required for the prokaryotic initiation of the translation). This protein has no significant homology to any known ribonuclease and its structure has never been investigated. The extreme toxicity of this ribonuclease prevents the expression of large quantities for structural studies. Here, we show that the toxicity of RegB can be bypassed by using the RegB H48A point mutant and explain why resolving the structure of this mutant is relevant. For nuclear magnetic resonance (NMR) purposes, we report the preparation of highly pure (13)C/(15)N double-labelled 1.2mM samples of RegB H48A using a high yield expression procedure in minimal medium (30 mg/L). We also present a set of solution conditions that maintain the concentrated samples of this protein stable for long periods at the NMR-required temperature. Finally, we present the first (1)H/(15)N and (1)H/(13)C two-dimensional NMR spectra of RegB H48A. These spectra show that the protein is folded and that the full structural analysis of RegB by NMR is feasible.

Solution NMR study of the monomeric form of p13suc1 protein sheds light on the hinge region determining the affinity for a phosphorylated substrate.

Cyclin-dependent kinase subunit (CKS) proteins bind to cyclin-dependent kinases and target various proteins to phosphorylation and proteolysis during cell division. Crystal structures showed that CKS can exist both in a closed monomeric conformation when bound to the kinase and in an inactive C-terminal beta-strand-exchanged conformation. With the exception of the hinge loop, however, both crystal structures are identical, and no new protein interface is formed in the dimer. Protein engineering studies have pinpointed the crucial role of the proline 90 residue of the p13(suc1) CKS protein from Schizosaccharomyces pombe in the monomer-dimer equilibrium and have led to the concept of a loaded molecular spring of the beta-hinge motif. Mutation of this hinge proline into an alanine stabilizes the protein and prevents the occurrence of swapping. However, other mutations further away from the hinge as well as ligand binding can equally shift the equilibrium between monomer and dimer. To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S. pombe p13(suc1) and its hinge mutant P90A in solution by NMR spectroscopy. We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding. Our structural study further indicates that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal beta-sheet involved in domain swapping and protein interactions.