Expression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the protegrin-3 precursor

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Etude structurale et biochimique de la proséquence de la protégrine

Chez le mammifères, de nombreux peptides antibactériens de séquences diverses possèdent une proséquence fortement homologue appelée motif cathélicidine. On peut supposer qu'elle intervient dans le transport et / ou dans le stockage d'antibactériens inactifs prêts à être libérés lors d'une agression bactérienne. Les protégrines ( PG, 16-18 résidus, 2 ponts disulfures), peptides antibactériens à large spectre d'activité, sont produites à partir d'un précurseur inactif composé d'un peptide signal, du motif cathélicidine ( ProS,.30-130) et de PG (131-147/148). Leur libération se fait par clivage de la liaison V130 - R131 par l'élastase. Bien que largement répandue, la structure 3D du motif cathélicidine (104-114 résidus, 2 ponts disulfures) n'étant pas connue. L'objectif du projet vise donc à déterminer la structure de ProS. Pour cela, un vecteur d'expression de ProS dans E.coli a d'abord été mis au point, pour ensuite produire la protéine native et uniformément marquée ( N15 et N15/C13) en quantité suffisante pour entreprendre son étude structurale à la fois par RMN et par radiocristallograohie. L'étude RMN a révélé pour ProS un repliement de type cystatine, composé d'une hélice N-terminale entourée par un feuillet beta constitué de 4 brins antiparallèles. Cette étude à également révélé une isomérie cis-trans des liaisons amides R87-P88 et D118-P119 donnant lieu à 4 conformères en échange lent dont la population dépend du PH. Par cristallographie, grâce à une stratégie originale qui consiste à remplacer les cystéines par des sélénocystéines et ainsi permettre l'utilisation des techniques MAD, la structure en solution a été confirmée. De plus, il a été montré que ProS dimérise par échange de domaine. Cette aptitude semble être un caractéristique du repliement de type cystatine. L'obtention de la structure de ProS devrait contribuer à mieux comprendre son rôle dans le processus de maturation des peptides antibactériensMONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

The [Lys-2-Arg-1-des(17−21)]-Endothelin-1 Peptide Retains the Specific Arg-1−Asp8 Salt Bridge but Reveals Discrepancies between NMR Data and Molecular Dynamics Simulations

The [des(17−21)]-endothelin-1 (CSH-ET) and [Lys-2-Arg-1-des(17−21)]-endothelin-1 (KR-CSH-ET) peptides, designed by removing the five-residue hydrophobic tail from the endothelin-1 (ET-1) and [Lys-2-Arg-1]-endothelin-1 (KR-ET-1) peptides, respectively, were synthesized. Previous studies on KR-ET-1 showed that, in contrast to ET-1, this engineered compound displays a pH-dependent conformational change related to the formation of a stabilizing salt bridge between the Arg-1 and Asp8 side chains. CD and NMR spectra indicate that CSH-ET and KR-CSH-ET display conformational behavior similar to those of ET-1 and KR-ET-1, respectively. The short salt bridge-stabilized KR-CSH-ET peptide therefore appears to be an attractive elementary scaffold for drug design. The solution structure of the salt-bridged form of KR-CSH-ET was determined by NMR at pH 4.5 and is very similar to the corresponding form of the parent KR-ET-1 peptide. Molecular dynamics simulations of the salt-bridged form of KR-CSH-ET were performed using both the GB/SA implicit solvation scheme or an explicit solvation and the particle-mesh Ewald method for long-range electrostatic calculation. Unexpectedly, the Arg-1−Asp8 salt bridge does not display in the simulation the stability that could be expected from the experimental data. The cooperative involvement of a cation−π interaction in formation of the salt bridge has been hypothesized. Difficulties in accurately simulating cation−π interactions might be responsible for the lack of stability in the simulation. At this time, however, no definitive explanation for the observed discrepancy between experiments and simulations is available, and further experimental studies appear to be necessary to fully understand in atomic detail the pH-dependent conformational change observed in the KR-ET-1 serie

Structure of the Cathelicidin Motif of Protegrin-3 Precursor: Structural Insights into the Activation Mechanism of an Antimicrobial Protein

International audienceCathelicidins are a family of antimicrobial proteins isolated from leucocytes and epithelia cells that contribute to the innate host defense mechanisms in mammalians. Located in the C-terminal part of the holoprotein, the cathelicidin-derived antimicrobial peptide is liberated by a specific protease cleavage. Here, we report the X-ray structure of the cathelicidin motif of protegrin-3 solved by MAD phasing using the selenocysteine-labeled protein. Its overall structure represents a fold homologous to the cystatin family and adopts two native states, a monomer, and a domain-swapped dimer. This crystal structure is the first example of a structural characterization of the highly conserved cathelicidin motif and thus provides insights into the possible mechanism of activation of the antimicrobial protegrin peptide

Solution Structure of the Recombinant Penaeidin-3, a Shrimp Antimicrobial Peptide

International audiencePenaeidins are a family of antimicrobial peptides of 47-63 residues isolated from several species of shrimp. These peptides display a proline-rich domain (N-terminal part) and a cysteine-rich domain (C-terminal part) stabilized by three conserved disulfide bonds whose arrangement has not yet been characterized. The recombinant penaeidin-3a of Litopenaeus vannamei (63 residues) and its [T8A]-Pen-3a analogue were produced in Saccharomyces cerevisiae and showed similar antimicrobial activity. The solution structure of the [T8A]-Pen-3a analogue was determined by using two-dimensional 1H NMR and simulated annealing calculations. The proline-rich domain, spanning residues 1-28 was found to be unconstrained. In contrast, the cysteine-rich domain, spanning residues 29-58, displays a well defined structure, which consists of an amphipathic helix (41-50) linked to the upstream and the downstream coils by two disulfide bonds (Cys32-Cys47 and Cys48-Cys55). These two coils are in turn linked together by the third disulfide bond (Cys36-Cys54). Such a disulfide bond packing, which is in agreement with the analysis of trypsin digests by ESI-MS, contributes to the highly hydrophobic core. Side chains of Arg45 and Arg50, which belong to the helix, and side chains of Arg37 and Arg53, which belong to the upstream and the downstream coils, are located in two opposite parts of this globular and compact structure. The environment of these positively charged residues, either by hydrophobic clusters at the surface of the cysteine-rich domain or by sequential hydrophobic residues in the unconstrained proline-rich domain, gives rise to the amphipathic character required for antimicrobial peptides. We hypothesize that the antimicrobial activity of penaeidins can be explained by a cooperative effect between the proline-rich and cysteine-rich features simultaneously present in their sequences

1XV3 : NMR structure of the synthetic penaeidin 4

Experimental Technique/Method:SOLUTION NMR Resolution: Classification:ANTIBIOTIC Release Date:2005-02-15 Deposition Date:2004-10-27 Revision Date:2008-04-30#2011-07-13 Molecular Weight:5313.24 Macromolecule Type:Protein Residue Count:47 Atom Site Count:368 DOI:10.2210/pdb1xv3/pdb Abstract: Antimicrobial peptide structure has direct implications for the complexity of functions and mechanisms of action. The penaeidin antimicrobial peptide family from shrimp is divided into multiple class designations based on primary structure. The penaeidin classes are not only characterized by variability in primary sequence but also by variation in target specificity and effectiveness. Whereas class 4 exhibits low isoform diversity within species and is highly conserved between species, the primary sequence of penaeidin class 3 is less conserved between species and exhibits considerable isoform diversity within species. All penaeidins, regardless of class or species, are composed of two dramatically different domains: an unconstrained proline-rich domain and a disulfide bond-stabilized cysteine-rich domain. The proline-rich domain varies in length and is generally less conserved, whereas the spacing and specific residue content of the cysteine-rich domain is more conserved. The structure of the synthetic penaeidin class 4 (PEN4-1) from Litopenaeus setiferus was analyzed using several approaches, including chemical mapping of disulfide bonds, circular dichroism analysis of secondary structural characteristics, and complete characterization of the solution structure of the peptide by proton NMR. L. setiferus PEN4-1 was then compared with the previously characterized structure of penaeidin class 3 from Litopenaeus vannamei. Moreover, the specificity of these antimicrobial peptides was examined through direct comparison of activity against a panel of microbes. The penaeidin classes differ in microbial target specificity, which correlates to variability in specific domain sequence. However, the tertiary structure of the cysteine-rich domain and indeed the overall structure of penaeidins are conserved across classes

Selenomethionine and Selenocysteine Double Labeling Strategy for Crystallographic Phasing

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