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

Abstract

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.

Epistatic effects between amino acid insertions and substitutions mediate toxin-resistance of vertebrate Na+,K+-ATPases

“…Resistance-conferring amino acid substitutions have identified in paralogs A1-A3, but are most common in ATP1A1, which is also the most ubiquitously expressed (Ujvari et al 2013;Ujvari et al 2015;Mohammadi et al 2016;Marshall et al 2018;Mohammadi et al 2022).…”

“…The evolution of toxin resistance in animals is among the best studied examples of adaptive molecular evolution (Brodie 2009). In many cases, diverse animals have convergently evolved resistance to the same toxin, allowing one to examine the extent to which genetic background and other factors constrain the process of adaptive protein evolution (McGlothlin et al 2016; Mohammadi et al 2022). Recent studies have highlighted how the potentially adaptive effects of particular amino acid mutations can strongly depend on the protein sequence background on which they arise (Weinreich et al 2006; Gong and Bloom 2014; Storz 2018).…”

“…Convergent evolution of resistance to CTS in diverse animals is often mediated, at least in part, by the evolution of NKA ‘target-site insensitivity’. Experimental studies have revealed a surprising consistency in the underlying molecular mechanisms of CTS insensitivity of NKA (Ujvari et al 2015; Storz 2016; Karageorgi et al 2019; Taverner et al 2019; Mohammadi et al 2021; Mohammadi et al 2022). Different combinations of amino-acid substitutions have been reported in animals that have evolved CTS-resistance, and available evidence suggests that some substitutions impede CTS binding by preventing the formation of hydrogen bonds between hydroxyl groups of CTS and polar residues of the H1-H2 extracellular loop (Laursen et al 2015).…”

“…In tetrapods, different isoforms of the NKA α-subunit are encoded by three paralogous members of a multigene family (ATP1A1-ATP1A3), with a fourth paralog specific to mammals (ATP1A4). Resistance-conferring amino acid substitutions have identified in paralogs A1-A3, but are most common in ATP1A1, which is also the most ubiquitously expressed (Ujvari et al 2013; Ujvari et al 2015; Mohammadi et al 2016; Marshall et al 2018; Mohammadi et al 2022).…”

“…In taxa as diverse as milkweed-feeding insects and toad-eating vertebrates, the evolution of CTS resistance is often associated with amino acid substitutions at sites 111 and 122 in the H1-H2 extracellular loop of ATP1A (Price and Lingrel 1988; Dobler et al 2012; Zhen et al 2012; Groen and Whiteman 2021; Mohammadi et al 2021; Mohammadi et al 2022). Consequently, these two sites have been the focus of experimental efforts to characterize molecular mechanisms of target-site insensitivity in NKA.…”