IDENTIFYING LIGAND/RECEPTOR INTERACTIONS IN A RESIDUE-LEVEL RESOLUTION THROUGH A COMBINATION OF EXPERIMENTAL AND IN SILICO MUTAGENESIS ANALYSIS

The molecular interactions between stem cell factor (SCF) and c-Kit receptor tyrosine kinase play a key role in hematopoiesis, fertility and in cancer development. Identification of residues most crucial for binding of the SCF/c-Kit complex is not only important for understanding of the evolution of its inter-molecular interactions but also for the design of new therapeutic molecules directed at inhibiting these interactions.

Experimentally, identification of such residues (i.e., epitope mapping) could be performed using alanine scanning mutagenesis, a process that is time consuming and laborious. Alternative methods based on various display and screening technologies allow exploration of all possible mutations and qualitative mapping of each individual residue contribution to binding affinity.

Herein, we used combinatorial single-mutation libraries to generate protein diversity, and yeast surface display (YSD) as a quantitative selection method to map the SCF/c-Kit binding epitope. The effects of mutations found in the four sub-domains (i.e., interface, surface, dimerization and core) of SCF were explained through experimental and computational structural analysis, based on the modeled structure of the complex. The SCF protein was much more destabilized by mutations in the core than by mutations in the surface. Computational and experimental results of the effects of mutations on the binding affinity were in good agreement and showed that most of the mutations that reduced affinity without structural disruption were localized to the SCF/c-Kit binding and SCF-SCF dimerization interfaces.

Identifying these buried residues might enable the development of target-specific therapeutic agents with ability to sterically occlude SCF/c-Kit binding interface, and the non-buried (surface and core) SCF residues, may aid in the understanding of SCF/c-Kit mechanism of interaction and further unravel new targets for SCF inhibition.