COMBINATORIAL AND COMPUTATIONAL APPROACHES TO IDENTIFY IMPORTANT RESIDUES IN THE M-CSF\C-FMS INTERACTION

The molecular interactions between macrophage colony stimulating factor (M-CSF) and the receptor c-fms play a key role in the immune response, bone metabolism and development of some cancers. Since 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 essential residues 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-CSF_M) single mutants and screened it to isolate variants with reduced affinity for c-fms using FACS. Sequencing yielded variants with mutations both in the direct binding interface and distant from it. In addition, we used computational modeling to map the identified mutations onto the M-CSF_M structure and classify them into three groups—those that significantly decrease protein stability, destroy favorable intermolecular interactions, or decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSF_M and three variants were produced as soluble proteins, and their affinity and structure were analyzed; good correlations with both YSD data and computational predictions were obtained. Next, we aim to crystallize M-CSF_M/c-fms complex. Crystallization of M-CSF_M revealed that in high concentrations, non-covalent interactions still enable the formation of dimers. Therefore, we wish to generate a variant in which those interactions are eliminated in order to understand the influence of dimerization on M-CSF structure and its biological activity.

By identifying the M-CSF_M residues critical for dimerization and for M-CSF/c-fms interactions, we lay down the basis for a deeper understanding of the M-CSF/c-fms signaling mechanism and development of target-specific therapeutic agents with ability to occlude M-CSF/c-fms binding interface.