COMBINATORIAL AND COMPUTATIONAL APPROCHES TO IDENTIFY THE M-CSF DIMERIZATION SITE INTERACTIONS

The molecular interactions between macrophage colony stimulating 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. M-CSF is a disulfide-linked homodimer that enables two c-fms units to come in close proximity and start their signaling cascade resulting in differentiation, survival and proliferation of macrophage precursor cells. In order to facilitate the design of drugs that inhibit M-CSF dimerization, it is important to establish which residues are critical for this process. Our goal was therefore to identify all the residues that are essential for human M-CSF self-association. In previous studies, it has been shown that C31S mutation inhibits M-CSF dimerization via disruption of a disulfide bond formation, and subsequent c-fms phosphorylation. In this study, we generated this C31S mutation (M-CSF_M) resulting in crystals that show other non-covalent intermolecular interactions but the same dimer arrangement. In order to analyze these non-covalent interactions, we used computational modeling to map and identify mutations onto the M-CSF_M dimerization site unraveling important sites for hydrogen bonds and electrostatic interactions that contribute to M-CSF dimerization. The computational analysis corresponded with our previous results that identified several mutations in the dimerization site that showed reduced affinity to c-fms caused by reduced M-CSF dimerization. Further validation of the M-CSF_M dimerization interactions was done by Analytical Ultra Centrifugation (AUC). By identifying the M-CSF_M residues critical for M-CSF dimerization interactions, we have laid down the basis for a deeper understanding of the M-CSF and other self-association mechanisms in protein and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF/c-fms signaling.