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

Abstract

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-CSF 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-CSF 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-CSF 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-CSF, 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-CSF 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 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.

Residue‐level determinants of angiopoietin‐2 interactions with its receptor Tie2

“…For example, 53 of the 183 residues in the Ang2‐BD and 42 of the 188 residues in the Tie2 ligand‐binding domain are within 10 Å of the Ang2‐Tie2 interface. To pinpoint the subset of Ang2 residues that actually contribute to interactions with Tie2 directly, we followed the approach we developed in previous studies . We applied the FDPB method to calculate the net electrostatic and polar contributions (ΔΔG elec ) of each Ang2 and Tie2 residue that is within 15 Å of the binding partner.…”

“…For a wider perspective, we applied our computational analysis to the SCF‐c‐Kit and M‐CSF‐c‐FMS complexes and compared them to previous random mutagenesis and YSD screening . Our comparison shows that our combined approach rapidly classified which RTK ligand residues contribute to receptor binding directly and which residues are sufficiently buried in the protein core to affect binding indirectly upon mutagenesis.…”

“…We followed the methodology described previously to analyze the per‐residue contributions of Ang2 and Tie2 residues to intermolecular interactions in the complex (Supporting Information Figure S1). The finite difference Poisson‐Boltzmann (FDPB) method was used to calculate the net electrostatic and polar contributions (ΔΔG elec ) of each residue that is within 15 Å of the dimer interface.…”

“…We used the following three-dimensional (3D) structures in our analysis (with PDB codes): Ang2-Tie2 (2GY7), 17 SCF-c-Kit (2E9W), 34 murine CSF-FMS (3EJJ), 35 human CSF (5LXF). 36 In our calculations, we used the individual domains of the ligand and the receptor that form the actual ligand-receptor complex, based on visual inspection of the structures: Ang2 313-495 (Ang2-BD), Tie2 23-210, SCF 2-140, and CSF 4-148. 3D structural visualization and superimpositions were carried out with the molecular graphics program Pymol (http:// pymol.org).…”

“…We followed the methodology described previously 32,[36][37][38][39][40] to analyze the per-residue contributions of Ang2 and Tie2 residues to intermolecular interactions in the complex (Supporting Information Figure S1).…”