Title
Identifying residues that determine SCF molecular-level interactions through a combination of experimental and in silicoanalyses
Author
Eitan Rabinovich, Michael Heyne, Anna Bakhman, Mickey Kosloff, Julia M. Shifman, Niv Papo
Year
2016
Journal
Journal of Molecular Biology
Abstract
The stem cell factor (SCF)/c-Kit receptor tyrosine kinase complex – with its significant roles in hematopoiesis and angiogenesis – is an attractive target for rational drug design. There is thus a need to map, in detail, the SCF/c-Kit interaction sites and the mechanisms that modulate this interaction. While most residues in the direct SCF/c-Kit binding interface can be identified from the existing crystal structure of the complex, other residues that affect binding through protein unfolding, intermolecular interactions, or allosteric or long-distance electrostatic effects cannot be directly inferred. Here, we describe an efficient method for protein-wide epitope mapping using yeast surface display (YSD). A library of single SCF mutants that span the SCF sequence was screened for decreased affinity to soluble c-Kit. Sequencing of selected clones allowed identification of mutations that reduce SCF binding affinity to c-Kit. Moreover, screening of these SCF clones for binding to a structural antibody, helped to identify mutations that result in small or large conformational changes in SCF. Computational modeling of the experimentally identified mutations showed that these mutations reduced the binding affinity through one of three scenarios: through SCF destabilization, through elimination of favorable SCF/c-Kit intermolecular interactions, or through allosteric changes. Eight SCF variants were expressed and purified. Experimentally measured in vitro binding affinities of these mutants to c-Kit confirmed both the YSD selection results and the computational predictions. This study has thus identified the residues crucial for c-Kit/SCF binding and has demonstrated the advantages of using a combination of computational and combinatorial methods for epitope mapping.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Biochemistry