INITIAL INSIGHTS INTO THE STRUCTURE-ACTIVITY RELATIONSHIPS OF AVIAN DEFENSINS.

Numerous β-defensins have been identified in birds and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian β-defensins (AvBDs), and the present work was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D- proteins clearly indicates that there is no chiral partner. Therefore the bacterial membrane is in all likelihood the primary target. Moreover, this work evidences that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for Gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural 3-stranded antiparallel β-sheet characteristic of β-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of avian β-defensin's family was analyzed. Well conserved residues were highlighted and the potential strategic role of the lysine 31 residue of AvBD2 emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity

Discovery and characterisation of a novel toxin from Dendroaspis angusticeps, named Tx7335, that activates the potassium channel KcsA

Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification of its disulphide-bridge scaffold. The toxin induces a dose-dependent increase in both open probabilities and mean open times on KcsA in artificial bilayers. Thus, it unexpectedly behaves as a channel activator rather than an inhibitor. A charybdotoxinsensitive mutant of KcsA exhibits similar susceptibility to Tx7335 as wild-type, indicating that the binding site for Tx7335 is distinct from that of canonical pore-blocker toxins. Based on the extracellular location of the toxin binding site (far away from the intracellular pH gate), we propose that Tx7335 increases potassium flow through KcsA by allosterically reducing inactivation of the channel

Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Conclusions/Significance Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.METASU

Assessing chemical labeling of proteins by mass spectrometry: MALDI−TOF to the rescue of ESI−HRMS

International audienc

Search for a high-mass calibrant for NALIM (Native Liquid MALDI MS): the case of mouse IgA

International audienceNative mass spectrometry (native MS) is a set of methods to analyze biomolecular assemblies directly in an instrument. To this end, biomolecular objects or complexes are exposed only to non-denaturing conditions to preserve their three-dimensional structure intact from the sample solution to the gas phase.In pharmacology, membrane proteins represent two-thirds of therapeutic targets, yet only 10% have been targeted so far. The characterization of membrane proteins by native MS can open many paths for biology and pre-clinical studies.To this day, the go-to method for native MS is based on electrospray ionization (ESI). However, membrane protein analysis by native ESI-MS is still a challenge. This is because these proteins necessitate detergents for solubilization in MS-compatible buffers. Detergents in turn can give rise to ion suppression, adduction, and degraded instrument performance. These effects can be somewhat mitigated by working above the detergent's critical micellar concentration [1].MALDI (Matrix-assisted laser desorption-ionization) ionization presents numerous advantages in this context. It is highly tolerant to contaminants and consumes little quantities of sample, making it de facto attractive for the native MS analysis of membrane proteins. Using these unique properties, we established a new native MS MALDI method using liquid spots called Native Liquid MALDI (NALIM) [2]. The success of this method relies on: (i) a matrix mixture that can be used in native conditions (pH 5), (ii) a liquid matrix that enables to gently transfer complexes in the gas phase while avoiding the transition through the solid state, which is the basis of classical solid-spot deposition methods. We are currently extending applications of the NALIM method to the observation of membrane protein complexes. In NALIM, we mainly observe low charge states (1+ and 2+), the monocharged state being largely predominant. Thus, the application of NALIM to large membrane protein-containing assemblies requires calibrant standards at high m/z ratios. In search of such calibrants, we investigated the use of an immunoglobulin A. Here we report on the suitability and usability of an IgA for calibration over a wide mass range. Optimizations include biochemical preparation, control of the degree of oligomerization and instrumental setup for NaLiM.[1] Barrera, N.P., Bartolo, N.D., Booth, P.J., and Robinson, C.V. (2008). Micelles Protect Membrane Complexes from Solution to Vacuum. Science 321, 243–246.[2] Beaufour, M., Ginguené, D., Le Meur, R., Castaing, B., and Cadene, M. (2018). Liquid Native MALDI Mass Spectrometry for the Detection of Protein-Protein Complexes. J. Am. Soc. Mass Spectrom. 29, 1981–1994

Development of an original footprinting approach to localize non-covalent ligand binding sites

International audienc

Towards a faster localization of noncovalent ligand binding sites using an original footprinting method

International audienc

Demonstration of a two-step reaction mechanism for the inhibition of heparin-bound neutrophil elastase by alpha 1-proteinase inhibitor.

International audienceHeparin decreases the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor as a result of its strong binding to the enzyme. Here, we used the slow-binding kinetic approach to decide whether the enzyme-inhibitor interaction proceeds via a two-step mechanism and to identify the step that is affected by heparin. The inhibition kinetics was assessed under pseudo-first-order conditions using conventional or stopped-flow spectrophotometry. In the absence of heparin, the pseudo-first-order rate constant of inhibition increased linearly with the inhibitor concentration indicating that within the experimental concentration range (EI with kass = 10(7) M-1 s-1) or to a two-step reaction (E+I Ki*EI* k2-->EI with Ki* > 0.4 microM and k2 > 4 s-1). In the presence of heparin, the rate constant of inhibition varied hyperbolically with the inhibitor concentration, indicating that the inhibition is a two-step process with Ki* = 80 nM and K2 = 0.15 s-1. Thus, heparin has two opposite effects on the elastase + alpha 1-proteinase inhibitor interaction: it favors the association by decreasing Ki* but impairs it by decreasing k2. This rationalizes the previously demonstrated rate-depressing effect of the sulfated polymer. Heparin does not significantly alter the stability of the irreversible elastase-alpha 1-proteinase inhibitor complex

SSPaQ: a subtractive method for the parallel quantification of the degree of modification at every possible site of a protein

International audienc