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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Molecular Modeling, Design and Structural Studies of a NewClass of Peptide Inhibitors of Bacterial TopoisomerasesLuiz Carlos B. Barbosa, Saulo S. Garrido, Davi B. Delfino,Anderson Garcia, and Reinaldo MarchettoInstitute of Chemistry, UNESP – Univ Estadual Paulista, Department of Biochemistry andChemistry Technology, Araraquara, SP, 14.800-900, BrazilIntroductionThe ParD-ParE is a bacterial toxin-antitoxin system that contributes to plasmid stability bya mechanism that relies on the differential stability of toxin and antitoxin proteins. ParE (a12.1 kDa protein) is the toxic component of this system which targets the intracellularDNA gyrase [1], unique type IIA topoisomerase able to introduce negative supercoils intobacterial DNA. The scarce information about its structure, its interactions with the antitoxinand with its intracellular target, led us consider the ParD-ParE system as a model of studyfor the design of new peptide inhibitors of bacterial topoisomerases.Results and DiscussionUnlike others members of the RelE/ParE superfamily that inhibit the translation byinducing cleavage of mRNAs, ParE acts blocking the DNA gyrase activity [1]. Recently,we built a 3D Model for E. coli ParE toxin [2]. Structural prediction from ParE primarystructure using PSIPRED server showed the - - -- - - sequence as secondary structure, as inferredin the proposed 3D Model (Figure 1). As anapproach for structure-function studies and based onproposed Model, we have designed and synthesizedby solid-phase methodology a series of linearpeptides and studied their activity by supercoilingand relaxation assays. Recent studies have shownthat residues at the extreme C-terminus of ParE arenecessary for protein stability and toxicity [3]. Inthis context, for the peptides design, initially weFig. 1. Predicted 3-D Structure ofEscherichia coli ParE toxin.considered the residues P80 to S105 that contain thepredicted C-terminal β-sheet and -helix structures.After, we also included other nineteen residues (L61to A79) in the peptide design. So, from the naturalParE, ten peptide sequences were obtained and the predicted C-terminal secondarystructures were confirmed by FTIR analyses (Figure 2). The ability of the peptides toinhibit the supercoiling reaction of DNA gyrase and the relaxation reaction oftopoisomerase IV (Topo IV) was investigated by gel electrophoresis with an initialscreening at 37°C and 100 µmol.L -1 of synthetic peptides. EcParE3 (P80-R100), EcParE8(L61-S105) and EcParE10 (L61-F87) were selected as good inhibitors for both DNAgyrase and Topo IV enzymes.EcParE1 ************** ************* ************* ***LMARLSER ******EcParE2 ************** ************* ********FHER MDLMARLSER ******EcParE3 ************** ************P ALVVAIFHER MDLMARLSER ******EcParE4 ************** ************* ************* ***LMARLSER LNIESEcParE5 ************** ************* ********FHER MDLMARLSER LNIESEcParE6 ************** ************P ALVVAIFHER MDLMARLSER LNIESEcParE7 ***********HY VFCLPHGSAP ALVVAIFHER MDLMARLSER LNIESEcParE8 LRMIHCEHHY VFCLPHGSAP ALVVAIFHER MDLMARLSER LNIESEcParE9 ***********HY VFCLPHGSAP ALVVAIF**** ************* ******EcParE10 LRMIHCEHHY VFCLPHGSAP ALVVAIF**** ************* ******Fig. 2. Primary structure of synthetic peptides from natural ParE protein.482