Mapping discontinuous protein‐binding sites via structure‐based peptide libraries: combining in silico and in vitro approaches |
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Authors: | Ines S Jaeger Ines Kretzschmar Jana Körner Armin A Weiser Carsten C Mahrenholz Ajish Potty Katerina Kourentzi Richard C Willson Rudolf Volkmer Robert Preissner |
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Institution: | 1. Institute for Physiology, Structural Bioinformatics Group, Charité‐Universit?tsmedizin Berlin, , 13125 Berlin, Germany;2. Institut für Medizinische Immunologie, Molecular Libraries and Recognition Group, Charité‐Universit?tsmedizin Berlin, , 10115 Berlin, Germany;3. Leibniz‐Institut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP), , 13125 Berlin, Germany;4. Systems Immunology Group, Institute for Theoretical Biology, Humboldt University, , Berlin, Germany;5. Research Center ImmunoSciences (RCIS) Hessische Strasse 3–4, , 10115 Berlin, Germany;6. EMD Millipore, , Bedford, MA, 01730 USA;7. University of Houston, Department of Chemical and Biomolecular Engineering, , Houston, TX, 77204‐4004 USA |
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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. Copyright © 2012 John Wiley & Sons, Ltd. |
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Keywords: | SPOT synthesis protein– protein interaction peptide library epitope lysozyme antibody |
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