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

Abstract

Comparative genomic studies reveal a global decline in rates of convergent amino acid substitution as a function of evolutionary distance. This pattern has been attributed to epistatic constraints on protein evolution, the idea being that mutations tend to confer the same fitness effects on more similar genetic backgrounds, so convergent substitutions are more likely to occur in closely related species. However, this hypothesis lacks experimental validation. We tested this model in the context of the recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse groups of 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 ATP1A1 predicted to confer CTS resistance, we performed protein engineering experiments to test the functional consequences of introducing these substitutions onto divergent species backgrounds. While we find that substitutions at these sites can have substantial background-dependent effects on CTS resistance, we also find no evidence for background-dependent effects on protein activity. We further show that the magnitude of a substitution’s effect on activity does not depend on the overall extent of ATP1A1 sequence divergence between species. More generally, a global analysis of substitution patterns across ATP1A orthologs and paralogs reveals that the probability of convergent substitution protein-wide is not predicted by sequence divergence. Together, these findings suggest that intramolecular epistasis is not an important constraint on the evolution of ATP1A CTS resistance in tetrapods.Significance StatementIndividual amino acid residues within a protein work in concert to produce a functionally coherent structure that must be maintained even as orthologous proteins in different species diverge over time. Given this dependence, we expect identical mutations to have more similar effects on protein function in more closely related species. We tested this hypothesis by performing protein-engineering experiments on ATP1A, an enzyme mediating target-site insensitivity to cardiotonic steroids (CTS) in diverse animals. These experiments reveal that although the phenotypic effects of substitutions can sometimes be background-dependent, the magnitude of these effects does not correlate with ATP1A1 sequence divergence. This implies that the genetic background across the ATP1A protein does not strongly limit the evolution of CTS resistance in animals.

Defence mitigation by predators of chemically defended prey integrated over the predation sequence and across biological levels with a focus on cardiotonic steroids

“…Most vertebrates possess three paralogs of the NKA subunit α gene ( ATP1A1-3 ) that have tissue-specific expression profiles and are associated with distinct physiological roles. Most amino acid variation among species and paralogs is concentrated in the first extracellular loop (residues 111–122; H1–H2 loop), which makes up part of the CTS binding domain and shows clade- and paralog-specific patterns of variability but also shows remarkable patterns of convergence, parallelism and divergence [ 125 ]. Amino acid substitutions at sites 111 and 122 in particular have been found to be key in the evolution of TSI in insect and vertebrate species [ 21 ] and have evolved in snakes [ 63 , 126 ], frogs [ 127 , 128 ] and other vertebrates [ 125 ].…”

“…[ 143 ]). In addition, they may possess altered target sites that are no longer susceptible to the toxic action of CTS [ 125 ]. Some predators sequester CTS from their prey and defend themselves against their own predators (e.g.…”

“…Endowing a protein with a new function through mutation often incurs a cost, particularly with respect to the protein's original function [ 125 , 128 , 140 , 183 ]. Functional studies of TSI have repeatedly shown that resistance-conferring substitutions often carry substantial functional costs to the ATPase activity of NKA [ 125 , 128 ].…”

“…Endowing a protein with a new function through mutation often incurs a cost, particularly with respect to the protein's original function [ 125 , 128 , 140 , 183 ]. Functional studies of TSI have repeatedly shown that resistance-conferring substitutions often carry substantial functional costs to the ATPase activity of NKA [ 125 , 128 ]. These negative pleiotropic effects can have major implications at higher biological levels due to the vital role that NKAs have in the maintenance of physiological homeostasis.…”

Defence mitigation by predators of chemically defended prey integrated over the predation cycle and across biological levels

“…Most vertebrates possess three paralogs of the NKA subunit α gene (ATP1A1-3) that have tissue-specific expression profiles and are associated with distinct physiological roles. Most amino acid variation among species and paralogs is concentrated in the H1-H2 extracellular loop (residues 111-122), which shows clade-and paralog-specific patterns of variability but also show remarkable patterns of convergence, parallelism, and divergence [130]. Amino acid substitutions at sites 111 and 122 in particular have been found to be key in the evolution of TSI in insect and vertebrate species [131] and have evolved in snakes [65,132], frogs [133,134], and other vertebrates [130].…”