Published: May 30, 2011 r2011 American Chemical Society 4627 dx.doi.org/10.1021/jm200311m | J. Med. Chem. 2011, 54, 4627–4637 ARTICLE pubs.acs.org/jmc Structure-Based Design of Short Peptide Ligands Binding onto the E. coli Processivity Ring Philippe Wolff, 9,† Vincent Oli  eric, 9,‡ Jean Paul Briand, § Olivier Chaloin, § Annick Dejaegere, || Philippe Dumas, † Eric Ennifar, † Gilles Guichard, ^ J  er^ ome Wagner, 4,# and Dominique Y. Burnouf * ,4,† † Architecture et R eactivit  e de l 0 ARN, Universit  e de Strasbourg, Institut de Biologie Mol  eculaire et Cellulaire, 15 rue Ren e Descartes, 67084, Strasbourg cedex, France ‡ Swiss Light Source (SLS), Paul-Scherrer-Institute (PSI), Villigen, Switzerland § CNRS, Institut de Biologie Mol  eculaire et Cellulaire, Immunologie et Chime Th erapeutiques, 15 rue Ren e Descartes, 67084, Strasbourg cedex, France ) IGBMC, D epartement de Biologie Structurale et G enomique, 1 rue Laurent Fries, BP10142, 67404 Illkirch, France ^ Institut Europ een de Chimie et Biologie, Universit  e de Bordeaux-CNRS UMR 5248, CBMN, 2, rue Robert Escarpit, 33607 Pessac, France # CNRS UMR7242, ESBS, Universit  e de Strasbourg, BP 10413, 67412 Strasbourg Cedex, France b S Supporting Information ’ INTRODUCTION In all three domains of life, multicomponent complexes, the so-called replisomes, have evolved to ensure the faithful replica- tion of chromosomal DNA. One central protein of these com- plexes forms a ring that encircles and slides along the double- stranded DNA. 1,2 A physical interaction between the clamp and the chromosomal replicase confers a high processivity to the enzyme. 3 In bacteria, the processivity factor, also referred to as the β clamp (hereafter named β), is a homodimer, which results from the head-to-tail association of two monomers, each of them being shaped in three globular subdomains. 1 In eukaryotes and archae, the β homologue factor, PCNA (for proliferating cell nuclear antigen), is a homotrimer with each monomer organized into two subdomains. 2,4 Besides their role as processivity factors for chromosomal replicases, β and PCNA clamps also participate in various proteinÀ protein interactions. They notably act as landing platforms for factors involved in DNA metabolism and cell cycle regulation, 5 particularly DNA polymerases involved in translesion synthesis, 6,7 and factors promoting DNA repair. 8À10 All these factors possess a small conserved peptide sequence, which binds into a hydro- phobic pocket located on one side of the clamp. It is noteworthy that these pockets differ significantly between bacterial and eukaryotic clamps. A bioinformatics analysis performed on putative β partners led to defining the bacterial consensus binding peptide QL[S/D]LF. 10 The absolute requirement of the interacting peptide for β clamp partners binding has been further demonstrated biochemically and physiologically. 11À14 Finally, the interaction between the clamp and the peptide of dif- ferent β binding proteins has been structurally characterized. 15À18 The peptide binding site is formed by a deep leucine-rich hydrophobic pocket (subsite 1) located between subdomains two and three of the β monomer and connected via a groove to a second subsite (subsite 2) located in subdomain 3 17 (Figure 1C). An additional interaction has also been observed in the case of Received: March 18, 2011 ABSTRACT: The multimeric DNA sliding clamps confer high pro- cessivity to replicative DNA polymerases and are also binding platforms for various enzymes involved in DNA metabolism. These enzymes interact with the clamp through a small peptide that binds into a hydrophobic pocket which is a potential target for the development of new antibacterial compounds. Starting from a generic heptapeptide, we used a structure-based strategy to improve the design of new peptide ligands. Chemical modifications at specific residues result in a dramatic increase of the interaction as measured by SPR and ITC. The affinity of our best hits was improved by 2 orders of magnitude as compared to the natural ligand, reaching 10 À8 M range. The molecular basis of the interactions was analyzed by solving the co-crystal structures of the most relevant peptides bound to the clamp and reveals how chemical modifications establish new contacts and contributes to an increased affinity of the ligand.