Structure of macrophage colony stimulating factor bound to FMS: diverse signaling assemblies of class III receptor tyrosine kinases

Abstract

Macrophage colony stimulating factor (M-CSF), through binding to its receptor FMS, a class III receptor tyrosine kinase (RTK), regulates the development and function of mononuclear phagocytes, and plays important roles in innate immunity, cancer and inflammation. We report a 2.4 A crystal structure of M-CSF bound to the first 3 domains (D1-D3) of FMS. The ligand binding mode of FMS is surprisingly different from KIT, another class III RTK, in which the major ligand-binding domain of FMS, D2, uses the CD and EF loops, but not the beta-sheet on the opposite side of the Ig domain as in KIT, to bind ligand. Calorimetric data indicate that M-CSF cannot dimerize FMS without receptor-receptor interactions mediated by FMS domains D4 and D5. Consistently, the structure contains only 1 FMS-D1-D3 molecule bound to a M-CSF dimer, due to a weak, hydrophilic M-CSF:FMS interface, and probably a conformational change of the M-CSF dimer in which binding to the second site is rendered unfavorable by FMS binding at the first site. The partial, intermediate complex suggests that FMS may be activated in two steps, with the initial engagement step distinct from the subsequent dimerization/activation step. Hence, the formation of signaling class III RTK complexes can be diverse, engaging various modes of ligand recognition and various mechanistic steps for dimerizing and activating receptors.

Fig. 1.

Stoichiometry and binding between M-CSF and FMS. (A) Gel filtration analysis of M-CSF, FMS, and the M-CSF:FMS complexes in a calibrated Superdex-200 column equilibrated and eluted with HBS buffer [10 mM Hepes (pH 7.5), 150 mM NaCl]. (Upper) Pink, M-CSF; green, FMS-D1–D3; lavender, FMS-D1-D5. (Lower) Red, M-CSF:FMS-D1–D3; blue, M-CSF:FMS-D1-D5; cyan, the 2:2 SCF:KIT complex and free SCF for comparison. (B) Thermodynamical profiles of M-CSF:FMS binding measured by calorimetry. M-CSF was injected into FMS-D1-D5 (Left) and FMS-D1–D3 (Right) in 2 separate experiments. The stoichiometry (N), affinity (K_d), enthalpy change (ΔH) and entropy change (ΔS) of each binding experiment are listed by the fitted curves.

Fig. 2.

Structure of the M-CSF:FMS-D1–D3 complex. (A) Ribbons representation of the complex, with the 2 M-CSF protomers in green and blue, and the FMS D1, D2, and D3 domains in orange, pink, and purple, respectively. The 2 N-linked glycans attached to FMS are depicted as sticks. (B) Surface representation of a completed, 2:2 M-CSF:FMS-D1–D3 complex in which the absent copy of FMS-D1–D3 (gray) is modeled based on the 2-fold symmetry between the 2 M-CSF protomers. (C) The different modes of ligand recognition between FMS and KIT as revealed by a comparison between M-CSF:FMS-D1–D3 (Left) and SCF:KIT-D1–D3 (Right) complexes. For clarity only one pair of receptor and ligand from each complex is shown. The ligands are in the same orientation. (D) Superimposition of FMS-D1-D2 and KIT-D1-D2 showing that D1 and D2 form an integral, rigid module.

Fig. 3.

The interaction between M-CSF and FMS. (A) An overview of the M-CSF:FMS interface, with FMS colored in pink and M-CSF in green. (B) GRASP surface potential models showing the charge distribution at the interface. (C) Close-up view of the site 1 interface between FMS-D2 and the M-CSF B and C helices. (D) Close-up view of the site 2 interface between FMS-D3 and the N-terminal segment of M-CSF.

Fig. 4.

The M-CSF conformational change upon FMS-D1–D3 binding. Comparison between the free M-CSF dimer (red) and the FMS-D1–D3-bound M-CSF dimer (green) shows that FMS-D1–D3 binding of 1 M-CSF protomer induces a rotational (5°) conformational change of the unoccupied M-CSF protomer.

Fig. 5.

A model for diverse dimerization and activation mechanisms of the class III RTKs. (A) Dimerization and activation are sequential events for KIT. KIT is dimerized directly by the high-affinity binding of SCF at the SCF:KIT interfaces, which in turn enables the membrane-proximal D4 and D5 domains to form secondary contact after a lateral movement. (B) Dimerization and activation are concurrent events for FMS. An M-CSF dimer recruits a single FMS in the first step. In the second step, the D4-D5 receptor-receptor interaction and the M-CSF:FMS interaction at the second site enable each other through enthalpic and entropic compensation, allowing the completion of a dimerized and activated complex. The FMS D1-D5 models were created by superimposing KIT D3-D5 (from PDB entry 2E9W) onto FMS D3 and then constructing a chimera of FMS-D1–D3 and the superimposed IT-D4-D5.