Benoit Odaert

Aggregation of Cateslytin β-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.

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.

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.

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

Cateslytin (bCGA 344RSMRLSFRARGYGFR358), 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 α-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 β-sheet contribution of almost 100% is detected at the air–water interface. Such conformational plasticity is discussed regarding the antimicrobial action of Cateslytin.

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.

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.

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.

Assignment of 1 H, 13 C and 15 N 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.

1 H, 13 C, 15 N 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.

CHAPTER 6:Magnetic Liposomes and Bicelles: New Tools for Membrane-Peptide Structural Studies

The structure and topology of membrane peptides and proteins in a natural membrane environment can be approached with solid-state NMR by making use of the magnetic properties of lipids, which in certain conditions lead to magnetically oriented membrane samples. Lipids possess an intrinsic very small magnetic susceptibility anisotropy, Δχ, which leads to interesting annealing properties in very high magnetic fields of ca. 20 Tesla. Saturated chain lipids have a negative Δχ, leading to liposome (multilamellar vesicle, MLV) deformation to prolate or to lipid bicelles (40–100 nm nanodiscs) oriented with the normal to the disc surface perpendicular to the field. Biphenyl chain-containing lipids exhibit a positive Δχ that leads to MLV oblate deformation or bicelle orientation with the normal to the nanodisc parallel to the field. Using different lipids either in the form of liposomes or bicelles to tune the membrane orientation with respect to the field, a collection of NMR experiments can be performed to gain membrane peptide/protein topology and structural information. Representative examples are given of the Pf1 coat membrane protein, the antimicrobial peptide surfactin and the neurotransmitter methionine enkephalin.