Differential Modes of Peptide Binding onto Replicative Sliding Clamps from Various Bacterial Origins Philippe Wolff, §,+ Ismail Amal, ¶,+ Vincent Olie ́ ric, ‡ Olivier Chaloin, # Gudrun Gygli, ¶,▼ Eric Ennifar, § Bernard Lorber, § Gilles Guichard, ○ Je ́ rôme Wagner, ∥ Annick Dejaegere, ¶ and Dominique Y. Burnouf* ,§ § Universite ́ de Strasbourg, UPR9002, Architecture et Ré activite ́ de l’ARN, Institut de Biologie Molé culaire et Cellulaire du CNRS, 15, rue Rene ́ Descartes, 67084 Strasbourg, France ¶ Institut de Ge ́ ne ́ tique et de Biologie Mole ́ culaire et Cellulaire (IGBMC), De ́ partement de Biologie Structurale et Ge ́ nomique, 1 rue Laurent Fries, BP10142, 67404 Illkirch, France ‡ Swiss Light Source (SLS), Paul-Scherrer-Institute (PSI), 5232 Villigen, Switzerland # Le Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Mole ́ culaire et Cellulaire, Laboratoire d'Immunopathologie et Chimie Thé rapeutiques, 15 rue Rene ́ Descartes, 67084 Strasbourg cedex, France ○ Universite ́ de Bordeaux, CNRS, IPB, UMR 5248, CBMN, Institut Europe ́ en de Chimie et de Biologie, 2 rue Robert Escarpit, 33607 Pessac, France ∥ CNRS UMR7242, ESBS, Universite ́ de Strasbourg, BP 10413, 67412 Strasbourg Cedex, France * S Supporting Information ABSTRACT: Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interact efficiently with our designed peptides and assemble the Escherichia coli and related orthologs clamps, whereas group II clamps poorly interact with the same peptides and include Bacillus subtilis and other Gram-positive clamps. These studies also suggest that the peptide binding process could occur via different mechanisms, which depend on the type of clamp. ■ INTRODUCTION The faithful replication of chromosomes is a major challenge for all organisms. For that purpose, they have evolved highly sophisticated mechanisms not only to copy the genetic material in an error-free manner but also to regulate the whole replicative process. 1 In Escherichia coli (Ec), the multisubunit DNA polymerase III completes the full chromosomal replication within 40 min under optimal growth conditions, at a rate of about 750 nucleotides/second. 2,3 Central to the efficiency of this process is the replicative processivity factor, also referred to as β ring or sliding clamp (SC), that anchors the polymerase onto DNA, thus conferring high processivity to the replicative enzyme. This homodimeric factor is loaded on DNA by the so-called γ complex in an ATP-dependent manner, slides rapidly along the double stranded helix, and interacts with the various polymerases (Pol I, II, III, IV, and V), 4 as well as with other proteins involved in DNA metabolism, such as MutS, Hda, or DNA ligase. 5−7 The interaction motif of all these proteins with SC was first identified using a bioinformatics approach. 5 This work identified a short interacting peptide in many eubacterial SC Received: February 28, 2014 Published: August 29, 2014 Article pubs.acs.org/jmc © 2014 American Chemical Society 7565 dx.doi.org/10.1021/jm500467a | J. Med. Chem. 2014, 57, 7565−7576