Protein interaction perturbation profiling at an amino acid resolution

Identification of genomic variants in healthy and diseased individuals continues to rapidly outpace our ability to functionally annotate them. As such, techniques that both systematically assay the functional consequences of nucleotide resolution variation and can scale to hundreds of genes are urgently required. To address this, we designed a sensitive yeast two-hybrid based “off switch” for positive selection of interaction disruptive variants from complex genetic pools. Combined with massively parallel programmed mutagenesis and a sequencing readout it enables systematic profiling of protein interaction determinants at an amino acid resolution. We defined >1,000 interaction disrupting amino acid mutations across eight subunits of the BBSome, the major human cilia protein complex associated with the pleiotropic genetic disorder Bardet-Biedl-Syndrome. These de novo high resolution interaction perturbation profiles provided a framework for interpreting patient derived mutations across the entire protein complex, highlighting how the impact of disease variation on interactome networks can be assessed systematically.