title: Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS.
abstract: The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF · c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSFM) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSFM variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSFM structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSFM and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSFM residues critical for M-CSF · c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF · c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.
authors.affiliation: From the Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, and.
authors.forename: Lior
authors.initials: L
authors.lastname: Rosenfeld
authors.affiliation: the Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
authors.forename: Jason
authors.initials: J
authors.lastname: Shirian
authors.affiliation: From the Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, and the Department of Physiology and Cell Biology, Ben-Gurion University of the Negev, Beer-Sheva 8410501 and.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Zur
authors.affiliation: the Department of Physiology and Cell Biology, Ben-Gurion University of the Negev, Beer-Sheva 8410501 and.
authors.forename: Noam
authors.initials: N
authors.lastname: Levaot
authors.affiliation: the Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Shifman
authors.affiliation: From the Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, and papo@bgu.ac.il.
authors.forename: Niv
authors.initials: N
authors.lastname: Papo
mesh_terms: D020650:Combinatorial Chemistry Techniques; D005434:Flow Cytometry; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000378:metabolism*
chemical_list: D016173:Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor
keywords: computer modeling; directed evolution; epitope mapping; ligand-binding protein; receptor tyrosine kinase