title: Identifying Residues that Determine SCF Molecular-Level Interactions through a Combination of Experimental and In silico Analyses.
abstract: The stem cell factor (SCF)/c-Kit receptor tyrosine kinase complex-with its significant roles in hematopoiesis and angiogenesis-is an attractive target for rational drug design. There is thus a need to map, in detail, the SCF/c-Kit interaction sites and the mechanisms that modulate this interaction. While most residues in the direct SCF/c-Kit binding interface can be identified from the existing crystal structure of the complex, other residues that affect binding through protein unfolding, intermolecular interactions, allosteric or long-distance electrostatic effects cannot be directly inferred. Here, we describe an efficient method for protein-wide epitope mapping using yeast surface display. A library of single SCF mutants that span the SCF sequence was screened for decreased affinity to soluble c-Kit. Sequencing of selected clones allowed the identification of mutations that reduce SCF binding affinity to c-Kit. Moreover, the screening of these SCF clones for binding to a structural antibody helped identify mutations that result in small or large conformational changes in SCF. Computational modeling of the experimentally identified mutations showed that these mutations reduced the binding affinity through one of the three scenarios: through SCF destabilization, through elimination of favorable SCF/c-Kit intermolecular interactions, or through allosteric changes. Eight SCF variants were expressed and purified. Experimentally measured in vitro binding affinities of these mutants to c-Kit confirmed both the yeast surface display selection results and the computational predictions. This study has thus identified the residues crucial for c-Kit/SCF binding and has demonstrated the advantages of using a combination of computational and combinatorial methods for epitope mapping.
authors.affiliation: Department of Biotechnology Engineering, and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Eitan
authors.initials: E
authors.lastname: Rabinovich
authors.affiliation: Department of Biotechnology Engineering, and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel; Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Michael
authors.initials: M
authors.lastname: Heyne
authors.affiliation: Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel.
authors.forename: Anna
authors.initials: A
authors.lastname: Bakhman
authors.affiliation: Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel.
authors.forename: Mickey
authors.initials: M
authors.lastname: Kosloff
authors.affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Shifman
authors.affiliation: Department of Biotechnology Engineering, and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Electronic address: papo@bgu.ac.il.
authors.forename: Niv
authors.initials: N
authors.lastname: Papo
mesh_terms: D061505:Cell Surface Display Techniques; D019295:Computational Biology; D004252:DNA Mutational Analysis; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D019089:Stem Cell Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit
keywords: binding affinity; combinatorial selection; computational protein design; protein engineering; protein–protein interactions