title: Mapping discontinuous protein-binding sites via structure-based peptide libraries: combining in silico and in vitro approaches.
abstract: To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three-dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein-protein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure-based approach and an experimental, high-throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate protein-protein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well-characterised murine IgG1 antibody HyHEL-5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL-5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme.
authors.affiliation: Institute for Physiology, Structural Bioinformatics Group, Charité-Universitätsmedizin Berlin, Lindenberger Weg 80, 13125 Berlin, Germany. ines.jaeger@charite.de
authors.forename: Ines S
authors.initials: IS
authors.lastname: Jaeger
authors.forename: Ines
authors.initials: I
authors.lastname: Kretzschmar
authors.forename: Jana
authors.initials: J
authors.lastname: Körner
authors.forename: Armin A
authors.initials: AA
authors.lastname: Weiser
authors.forename: Carsten C
authors.initials: CC
authors.lastname: Mahrenholz
authors.forename: Ajish
authors.initials: A
authors.lastname: Potty
authors.forename: Katerina
authors.initials: K
authors.lastname: Kourentzi
authors.forename: Richard C
authors.initials: RC
authors.lastname: Willson
authors.forename: Rudolf
authors.initials: R
authors.lastname: Volkmer
authors.forename: Robert
authors.initials: R
authors.lastname: Preissner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D019151:Peptide Library; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods; D011506:Proteins / Q000737:chemistry*; D012984:Software
chemical_list: D007074:Immunoglobulin G; D019151:Peptide Library; D011506:Proteins; C421864:hen egg lysozyme; D009113:Muramidase