MAPPING BINDING LANDSCAPES OF DIFFERENT TRYPSIN/BPTI COMPLEXES BY EXPERIMENTAL AND COMPUTATIONAL METHODS

Protein-protein interactions (PPIs) regulate many different functions in a cell, e.g. signal transduction, gene expression and cell growth. While molecular interactions important for the binding process have been studied for a long time, it is not well understood how the interplay of these interactions produces such large differences in PPI binding affinities even if one ligand binds to multiple targets. This study aims at mapping binding landscapes in three homologous complexes between trypsin-like proteins (human cationic trypsin, human mesotrypsin and bovine trypsin) and their inhibitor BPTI spanning over nine orders of magnitude. These binding interfaces are experimentally investigated by saturated mutagenesis meaning that all BPTI binding interface residues are individually mutated to all amino acids. The resulting binding to the different trypsins of all the constructed BPTI libraries is measured by FACS. High- and low-affinity mutants are selected by yeast surface display (YSD) and flow cytometry and sequenced. Sequencing analysis allows us to find motifs in the mutants responsible for distinct affinities. Furthermore, we are developing a computational approach that calculates affinities between mutated proteins allowing a fast and reliable analysis of the newly explored PPIs with known crystal structure. A comparison between computational and experimental results will help us to validate and improve the computational protocol. The proposed study addresses fundamental questions on PPI evolution such as what variability in affinity could be achieved by single mutations and how this variability differs between high- and low-affinity complexes. In addition, our results will pave the way for development of novel inhibitors for a particular kind of serine proteases.