Epistasis shapes the fitness landscape of an allosteric specificity switch

Epistasis is a major determinant in the emergence of novel protein function. In allosteric proteins, direct interactions between inducer-binding mutations propagate through the allosteric network, manifesting as epistasis at the level of biological function. Elucidating this relationship between local interactions and their global effects is essential to understanding evolution of allosteric proteins. We integrate computational design, structural and biophysical analysis to characterize the emergence of novel inducer specificity in an allosteric transcription factor. Adaptive landscapes of different inducers of the designed mutant show that a few strong epistatic interactions constrain the number of viable sequence pathways, revealing ridges in the fitness landscape leading to new specificity.

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Fig: Design of resveratrol-specific TtgR variant: Resveratrol conformers are docked into TtgR followed by Rosetta-based computational design of the binding pocket. Candidates with favorable Rosetta score metrics (green points) are synthesized and cloned into an expression vector. Distribution of fluorescence in cells containing uninduced TtgR variant library (light green), induced with naringenin (light blue) and resveratrol (red) before sorting (Pre-Sort) and after three rounds of sorting (Post-Sort) are shown. Colony screening identified a quadruple mutant showing resveratrol specificity: C137I/I141W/M167L/F168Y. The quadruple mutant phenotype was compared to wild-type in biological triplicate (n=3) by inducing each with either 1000 μM naringenin or 100 μM resveratrol. The error bars denote the standard deviation of the fold induction for the triplicate measurements (see “methods”).

The structure of the designed mutant shows that a striking change in inducer orientation still retains allosteric function. Comparing biophysical and functional properties suggests a nonlinear relationship between inducer binding affinity and allostery. Our results highlight the functional and evolutionary complexity of allosteric proteins.

Nishikawa, K.K., Hoppe, N., Smith, R. et al. Epistasis shapes the fitness landscape of an allosteric specificity switch. Nat Commun 12, 5562 (2021). https://doi.org/10.1038/s41467-021-25826-7

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