A Phage-Assisted Continuous Selection Approach for Deep Mutational Scanning of Protein-Protein Interactions

Abstract

Protein-protein interactions (PPIs) are critical for organizing molecules in a cell and mediating signaling pathways. Dysregulation of PPIs are often key drivers of disease. To better understand the biophysical basis of such disease processes – and to potentially target them - it is critical to understand the molecular determinants of PPIs. Deep mutational scanning (DMS) facilitates the acquisition of large amounts of biochemical data by coupling selection with high throughput sequencing (HTS). The challenging and labor-intensive design and optimization of a relevant selection platform for DMS, however, limits the use of powerful directed evolution and selection approaches. To address this limitation, we designed a versatile new phage assisted continuous selection (PACS) system using our proximity-dependent split RNA polymerase (RNAP) biosensors with the aim of greatly simplifying and streamlining the design of a new selection platform for PPIs. After characterization and validation using the model KRAS/RAF PPI, we generated a library of RAF variants and subjected them to PACS and DMS. Our HTS data revealed that amino acid (aa) positions 66, 84, and 89 on RAF, key residues in the KRAS/RAF PPI, are intolerant to mutations. We also identified a subset of residues with broad aa substitution tolerance, aa positions 52, 55, 76, and 79. Due to the plug and play nature of RNAP biosensors, this method can easily be extended to other PPIs. More broadly, this, and other methods under development, supports the application of evolutionary and high-throughput approaches to bear on biochemical problems, moving towards a more comprehensive understanding of sequence-function relationships in proteins.

Phage-DMS: a comprehensive method for fine mapping of antibody epitopes

“…Moreover, unlike methods that involve growth of the virus in culture, phage will display peptides encoding mutations irrespective of their effect on replication fitness, resulting in a more complete picture of the potential effect of all variants on binding. Given its versatility, Phage-DMS is not limited to mapping antibody epitopes and could theoretically be used to map binding sites between any two proteins of interest, similar to other studies that have utilized DMS to dissect protein-protein interactions using phage [10][11][12][13] .…”

“…We have built upon previous studies employing phage display in combination with DMS [10][11][12][13] and here we describe Phage-DMS, a new method that allows high-throughput and high-resolution mapping of antibody epitopes that are proximal in primary sequence with only a single round of immunoprecipitation. Using Phage-DMS, we identified the epitope of four well characterized HIV mAbs, confirming sites of escape predicted using other approaches as well as finding novel epitope sites.…”