School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, United States of America.
Molecular Evolutionary Biology, Institut für Zell- und Systembiologie der Tiere, Universität Hamburg, Hamburg, Germany.
PLoS Genet. 2022 Aug 16;18(8):e1010323. doi: 10.1371/journal.pgen.1010323. eCollection 2022 Aug.
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 (See S1 Text for Spanish translation).
越来越多的理论和实验证据表明，分子内上位性是蛋白质进化速率和模式的主要决定因素，对新蛋白质功能的进化施加了实质性的限制。在这里，我们研究了分子内上位性在四足动物中心脏毒素（CTS）抗性的反复进化中的作用，这种进化是通过 Na，K-ATP 酶（ATP1A）的α亚基家族的特定氨基酸取代发生的。在鉴定出一系列在 ATP1A 的两个关键位点发生的反复取代，这些取代可预测在各种四足动物中产生 CTS 抗性后，我们随后进行了蛋白质工程实验，以测试将这些取代引入不同物种背景下的功能后果。与之前的结果一致，我们发现这些位点的取代对 CTS 抗性有很大的背景依赖性影响。然而，从全局来看，这些取代也具有多效性，与背景依赖性影响一致，而不是加性影响。此外，取代对活性的影响程度不取决于物种之间 ATP1A 序列差异的总体程度。我们的研究结果表明，对 CTS 抗性 Na，K-ATP 酶进化的上位性限制可能取决于少数几个位点，而与蛋白质分化的总体水平几乎没有关系。我们提出，对有限数量的位点的依赖可能解释了在具有高度分化的 Na，K-ATP 酶的分类群中观察到的趋同 CTS 抗性取代的现象（有关西班牙语翻译，请参阅 S1 文本）。
