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 is often a key driver 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 previously reported 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 positions along the binding interface that are both tolerant and intolerant to mutations, as well as which substitutions are tolerated at each position. Critically, the “functional scores” obtained from enrichment data through continuous selection for individual variants correlated with K D values measured in vitro, indicating that biochemical data can be extrapolated from sequencing using our new system. Due to the plug and play nature of RNAP biosensors, this method can likely be extended to a variety of other PPIs. More broadly, this, and other methods under development support the continued development of evolutionary and high-throughput approaches to address biochemical problems, moving toward a more comprehensive understanding of sequence–function relationships in proteins.

Contingency and chance erase necessity in the experimental evolution of ancestral proteins

“…Cloned expression vectors contained the following: (1) a previously evolved, isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible N-terminal half of T7 RNAP ( Zinkus-Boltz et al, 2019 ) fused to a BCL-2 family protein; (2) the C-terminal half of T7 RNAP fused to a peptide from a BH3-only protein; and (3) T7 promoter-driven luciferase reporter. Chemically competent S1030 E. coli cells ( Carlson et al, 2014 ) were prepared by culturing to an OD 600 of 0.3, washing twice with a calcium chloride/HEPES solution (60 mM CaCl 2 , 10 mM HEPES pH 7.0, 15% glycerol), and then resuspending in the same solution.…”

“…Cloned expression vectors contained the following: 1) a previously-evolved, isopropyl β-D-1thiogalactopyranoside (IPTG)-inducible N-terminal half of T7 RNAP (Zinkus-Boltz et al, 2019) fused to a BCL-2 family protein; 2) the C-terminal half of T7 RNAP fused to a peptide from a BH3-only protein; and…”

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 ( Ernst et al., 2010 ; Fowler et al., 2010 ; Starita et al., 2013 ; Zinkus-Boltz et al., 2019 ).…”

“…We have built upon previous studies employing phage display in combination with DMS ( Ernst et al., 2010 ; Fowler et al., 2010 ; Starita et al., 2013 ; Zinkus-Boltz et al., 2019 ), 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.…”

Deep mutational scan of a drug efflux pump reveals its structure-function landscape

“…DMS allows studying the effect of hundreds of single variants with regards to protein activity in a single experiment by combining protein libraries with a selection procedure and NGS as a selection read-out [11]. DMS has thus far been mainly used to study protein-protein interactions and catalytic activity profiles of enzymes [12,13].…”