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.

A system for the evolution of protein-protein interaction inducers

“…This observation mirrored the T47A, A63E double mutant discovered in our evolution that demonstrated a small yet significant increase in LC3B binding when compared to the wildtype ULK1 peptide (Figure 5D). These data, along with the mammalian cell co-immunoprecipitation experiment and previous work 74,80,94 , support the theory that observed changes in activity translate beyond the split RNAP system to biologically relevant interactions.…”

“…During our validation of rePPI-G, we discovered affinity-altering mutations that parallel those found in previous studies. For example, our group used PACS to perform deep mutational scanning on the KRas/Raf1 interaction interface 80 . While most mutations in Raf1 resulted in either no change or a decrease in enrichment, N71K resulted in the most positive enrichment score, indicating this mutation increases the interaction affinity.…”

“…Critically, we demonstrated that the split T7 RNAP system can detect validated molecular glues, including both the rapamycin-induced pairing of FRB/FKBP 74,77 and the abscisic acid-induced interaction of PYL/ABI [78][79] . Moreover, we recently developed "Phage-Assisted Continuous Selection -Deep Mutational Scanning" (PACS-DMS), a method that links PPIs between a target protein and a library of binding variants to phage fitness, thereby allowing the interrogation of mutations that impact PPI formation 80 . Therefore, we hypothesized that the proximity dependent split RNAP system could be used to convert induced PPIs into gIII production, thus developing a new PACE system for evolving PPI inducers.…”

“…Although these methods can take months to develop and achieve suitable activity to begin a PACE experiment, they are excellent at amplifying rare variants from a large pool. We hypothesized that a short round of "Phage-Assisted Non-Continuous Selection" (PANCS), a combination of PANCE 91 and PACS 80 , could quickly amplify rare PACE variants capable of escaping the PPI inducer activity minima.…”

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.…”