Combinatorial and computational approaches to identify interactions of macrophage colony-stimulating factor (M-CSF) and its receptor c-FMS

Lior Rosenfeld, Jason Shirian, Yuval Zur, Noam Levaot, Julia M. Shifman, Niv Papo

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

The molecular interactions between macrophage colonystimulating 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.

Original languageEnglish
Pages (from-to)26180-26193
Number of pages14
JournalJournal of Biological Chemistry
Volume290
Issue number43
DOIs
StatePublished - 23 Oct 2015

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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