Pervasive degeneracy and epistasis in a protein-protein interface

Abstract

Signal transduction pathways rely on transient yet specific protein-protein interactions. How a limited set of amino acids can enforce cognate protein interactions while excluding undesired pairings remains poorly understood, even in cases where the contacting residues have been identified on both protein partners. To tackle this challenge, I performed structure-guided and library-based mutagenesis studies of bacterial two-component signaling pathways. These pathways, typically consisting of a histidine kinase and a response regulator, are an ideal model system for studying protein-protein interactions as they rely almost exclusively on molecular recognition for specificity. The kinase uses a limited set of residues to recognize the regulator in both phosphorylation and dephosphorylation reactions, and to prevent docking with all noncognate regulators. In this thesis I characterized the extent to which interface residues in two-component signaling proteins can be modified without changing the overall behavior of the pathway. In collaboration with another research group I have performed a mutagenesis study of a two-component system from Thermotoga maritima that has proven amenable to structural analysis. By solving the cocrystal structure of a histidine kinase and response regulator containing interface residues from a different interacting pair, we learned the biophysical basis for accommodating these new residues. To understand how many different residue combinations can support a functional interaction, I comprehensively mapped the sequence space of the interface formed by Escherichia coli histidine kinase PhoQ and its partner PhoP. I used a robust high-throughput assay to screen a library of 204 (160,000) PhoQ variants in which I had completely randomized the four key specificity-determining residues. Using deep sequencing, I identified -1,600 (1 %) variants that can phosphorylate and dephosphorylate PhoP as well as the wild-type PhoQ. Strikingly, PhoQ can interact with PhoP via many sets of interfacial residues that are completely different from the wild type. This combinatorial approach to mapping sequence space revealed interdependencies between individual amino acids, illustrating its power relative to screens that only examine substitutions at individual sites. This thesis provides a framework for mapping the sequence space of histidine kinases and has broad implications for understanding protein-protein interaction specificity and the evolution of bacterial signaling pathways.