Data from: Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence

A growing body of theoretical and experimental evidence suggests that
intramolecular epistasis is a major determinant of rates and patterns of
protein evolution and imposes a substantial constraint on the evolution of
novel protein functions. Here, we examine the role of intramolecular
epistasis in the recurrent evolution of resistance to cardiotonic steroids
(CTS) across tetrapods, which occurs via specific amino acid substitutions
to the α-subunit family of Na,K-ATPases (ATP1A). After identifying a
series of recurrent substitutions at two key sites of ATP1A that are
predicted to confer CTS resistance in diverse tetrapods, we then performed
protein engineering experiments to test the functional consequences of
introducing these substitutions onto divergent species backgrounds. In
line with previous results, we find that substitutions at these sites can
have substantial background-dependent effects on CTS resistance. Globally,
however, these substitutions also have pleiotropic effects that are
consistent with additive rather than background-dependent effects.
Moreover, the magnitude of a substitution’s effect on activity does not
depend on the overall extent of ATP1A sequence divergence between species.
Our results suggest that epistatic constraints on the evolution of
CTS-resistant forms of Na,K-ATPase likely depend on a small number of
sites, with little dependence on overall levels of protein divergence. We
propose that dependence on a limited number sites may account for the
observation of convergent CTS resistance substitutions observed among taxa
with highly divergent Na,K-ATPases.