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.

Analysis of Arabidopsis Accessions Hypersensitive to a Loss of Chloroplast Translation

“…Recent studies of human disease genes have examined deleterious variants that are fixed in the genomes of other vertebrates, often with a single epistatic substitution elsewhere in the protein (Jordan et al, 2015). Widespread epistasis has also been documented in large-scale gene replacement studies in microorganisms (Podgornaia and Laub, 2015;Li et al, 2016). Whether the unexpected tolerance of some ACCase variants is also context dependent, and would be reversed in a different sequence background, remains to be determined.…”

Enhanced identification of synergistic and antagonistic emergent interactions among three or more drugs

“…Importantly, this particular rescaling method is different from the rescaling previously used in the literature by ourselves and others. Consequently, this could reveal new insights for interactions among three or more drugs, as well as other objects such as proteins [22] and predators [23][24][25][26][27], and some existing results may need to be revisited and revised [14,28].…”

Information capacity of specific interactions

“…In 1894, Emil Fischer proposed the "lock and key" model as an analogy for understanding enzyme-substrate specificity (42), focusing on the physical shapes of paired interacting components; mutual information encompasses this idea and can be applicable to a large number of biological systems. In particular, our model is useful for predicting the differences between interacting proteins that use shape complementarity alone and those that combine both shape and electrostatic complementarity (e.g., Dpr-DIP vs. Dscam proteins) (43) and may also be applied to a host of other biological interaction networks (22) where information transmission and pair specificity play critical roles in biological function [e.g., histidine kinase/response regulator proteins (44) and the immune system (25)]. Crucially, the mutual information model provided above is flexible enough to be extended to some of the challenging physics encountered in biology.…”