A Directed Evolution Approach Selecting for Protein Complex-Interfering Mutations

Understanding the topologies and functions of protein interaction networks requires the selective removal of single interactions. We introduce an evolutionary approach that selects among a random library of alleles for mutations that impair the binding to a given partner protein. The selection makes use of a Split-Ubiquitin based protein interaction assay. This assay provides yeast cells that carry protein complex disturbing mutations with the advantage to survive on uracil-lacking media. Applied to the exemplary interaction between the PB domains of the yeast proteins Bem1 and Cdc24, we performed two independent selections. The selections were either analyzed by Sanger sequencing of isolated clones or by next generation sequencing (NGS) of pools of clones. Both screens enriched for the same mutation in position 833 of Cdc24. Biochemical analysis confirmed that this mutation disturbs the interaction with Bem1 but not the fold of the protein. The larger data set obtained by NGS achieved a more complete representation of the interaction interface of Cdc24.