Sequence-Function mapping of MKK1-ERK2 interactions reveals positive epistasis in the MKK1 D-domain. MKK1 D-domain

The combination of ultrahigh-throughput screening (uHTS) and deep sequencing represents a powerful approach to protein engineering, informing on function and underlying epistatic interactions within mutant protein libraries. However, human protein kinases remain under-explored by combinatorial sequence-function mapping, and current screens suffer from interference from functional redundancy in cells, limiting elucidation of the intricate fine-tuning of signalling cascades. Here we present an entirely in vitro uHTS approach (10^7 variants per day) for testing phosphate transfer in protein kinase pathways and cascades. Screening a 500,000-membered MKK1 library for functional complementarity to ERK2 revealed the MKK1 docking domain to be highly epistatic, combining an anchor motif of large hydrophobic residues with a more varied ensemble of epistatically linked amino acids to facilitate binding and phosphorylation of ERK2.