Engineering a monomeric variant of macrophage colony-stimulating factor (M-CSF) that antagonizes the c-FMS receptor

Yuval Zur, Lior Rosenfeld, Anna Bakhman, Stefan Ilic, Hezi Hayun, Anat Shahar, Barak Akabayov, Mickey Kosloff, Noam Levaot, Niv Papo

Research output: Contribution to journalArticlepeer-review


Enhanced activation of the signaling pathways that mediate the differentiation of mononuclear monocytes into osteoclasts is an underlying cause of several bone diseases and bone metastasis. In particular, dysregulation and overexpression of macrophage colony-stimulating factor (M-CSF) and its c-FMS tyrosine kinase receptor, proteins that are essential for osteoclast differentiation, are known to promote bone metastasis and osteoporosis, making both the ligand and its receptor attractive targets for therapeutic intervention. With this aim in mind, our starting point was the previously held concept that the potential of the M-CSFC31S mutant as a therapeutic is derived from its inability to dimerize and hence to act as an agonist. The current study showed, however, that dimerization is not abolished in M-CSFC31S and that the protein retains agonistic activity toward osteoclasts. To design an M-CSF mutant with diminished dimerization capabilities, we solved the crystal structure of the M-CSFC31S dimer complex and used structure-based energy calculations to identify the residues responsible for its dimeric form. We then used that analysis to develop M-CSFC31S,M27R, a ligand-based, high-affinity antagonist for c-FMS that retained its binding ability but prevented the ligand dimerization that leads to receptor dimerization and activation. The monomeric properties of M-CSFC31S,M27R were validated using dynamic light scattering and small-angle X-ray scattering analyses. It was shown that this mutant is a functional inhibitor of M-CSF-dependent c-FMS activation and osteoclast differentiation in vitro. Our study, therefore, provided insights into the sequence–structure–function relationships of the M-CSF/c-FMS interaction and of ligand/ receptor tyrosine kinase interactions in general.

Original languageEnglish
Pages (from-to)2601-2617
Number of pages17
JournalBiochemical Journal
Issue number15
StatePublished - 1 Aug 2017

Bibliographical note

Funding Information:
This work was supported by the European Research Council ?Ideas program? ERC-2013-StG [contract grant no. 336041] to N.P. N.L. acknowledges support by grant from the Israel Science Foundation [grant no. 544/15]. M.K. acknowledges support by grants from the Israel Science Foundation [grant nos 1454/13, 1959/13, and 2155/15] and from the Israel Ministry of Science, Technology and Space, Israel and the Italian Ministry of Foreign Affairs [3-10704]. The authors thank Dr Alon Zilka for his technical assistance. We thank the EMBL staff Dr Hassan Belrhali and Dr Babu A. Manjasetty for providing support on the beamline and EMBL-DBT for providing access to the BM14 beamline at the ESRF. Crystallization and structure determination were performed at the Macromolecular Crystallography Research Center (MCRC), NIBN, BGU. The DLS and SAXS experiments were conducted at the Ilse Katz Institute for Nanoscale Science and Technology with the help of Dr Sharon Hazan.

Publisher Copyright:
© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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