Disentangling contact and ensemble epistasis in a riboswitch
Mutations introduced into macromolecules often exhibit epistasis, where the effect of one mutation alters the effect of another. Knowledge of the mechanisms that lead to epistasis is important for understanding how macromolecules work and evolve, as well as for effective macromolecular engineering. Here we investigate the interplay between "contact epistasis" (epistasis arising from physical interactions between mutated residues) and "ensemble epistasis" (epistasis that occurs when a mutation redistributes the conformational ensemble of a macromolecule, thus changing the effect of the second mutation). We argue that the two mechanisms can be distinguished in allosteric macromolecules by measuring epistasis at differing allosteric effector concentrations. Contacts give rise to epistasis in the microscopic equilibrium constants describing the conformational ensemble. Ensemble epistasis manifests in thermodynamic observables, such as the energy of ligand binding or enzyme activation, that depend on the concentration of allosteric effector. Using this framework, we experimentally investigated the origins of epistasis in three mutant cycles introduced into the adenine riboswitch aptamer domain. We found evidence for both contact and ensemble epistasis in all cycles. Further, we found that the two mechanisms of epistasis can interact with each other. For example, in one mutant cycle we observe contact epistasis of 6 kcal/mol attenuated by the ensemble to only 1.5 kcal/mol in the final thermodynamic observable. Finally, our work yields simple heuristics for identifying contact and ensemble epistasis using limited experimental measurements. Statement of significanceMutations to protein or RNA molecules often have different effects when introduced individually versus together. To understand and engineer biological macromolecules, we must identify the mechanistic origins of this phenomenon. Here, we measured the interplay between direct, physical interactions between mutations ("contact epistasis") and indirect interactions mediated by conformational ensembles ("ensemble epistasis"). We introduced pairs of mutations into an RNA molecule that transitions between several different conformations. We found epistasis arising from both contacts and the ensemble, and that the two mechanisms could synergize with one another. Our work reveals that one must consider the effects of mutations on multiple conformations to understand epistasis and suggests a few rules-of-thumb for disentangling contact and ensemble epistasis in other macromolecules.