Abstract
Cation/proton antiporters (CPAs) play a major role in maintaining living cells’ homeostasis. CPAs are commonly divided into two main groups, CPA1 and CPA2, and are further characterized by two main phenotypes: ion selectivity and electrogenicity. However, tracing the evolutionary relationships of these transporters is challenging because of the high diversity within CPAs. Here, we conduct comprehensive evolutionary analysis of 6537 representative CPAs, describing the full complexity of their phylogeny, and revealing a sequence motif that appears to determine central phenotypic characteristics. In contrast to previous suggestions, we show that the CPA1/CPA2 division only partially correlates with electrogenicity. Our analysis further indicates two acidic residues in the binding site that carry the protons in electrogenic CPAs, and a polar residue in the unwound transmembrane helix 4 that determines ion selectivity. A rationally designed triple mutant successfully converted the electrogenic CPA, EcNhaA, to be electroneutral.
Highlights
Cation/proton antiporters (CPAs) play a major role in maintaining living cells’ homeostasis
Based on experiments with Escherichia coli NhaA (EcNhaA), it has been suggested that electrogenic CPAs are characterized by two conserved aspartates in their ion binding site—D163 and D164 on transmembrane helices (TMs)-5 in EcNhaA
The two different methods divided the CPA superfamily into the same main clades (Supplementary Fig. 2), and the results presented here are based on the IQ-TREE analysis
Summary
Cation/proton antiporters (CPAs) play a major role in maintaining living cells’ homeostasis. CPAs are often classified based on two phenotypes, their ion selectivity, i.e., whether they transport Na+ or K+, and their electrogenicity, i.e., whether a cation exchanges for one (electroneutral) or two (electrogenic) protons. A few studies have investigated the molecular basis for the ion selectivity of CPAs. Site directed mutagenesis of the sodium-selective SOD22 from Zygosaccharomyces rouxii (ZrSOD22) suggested that a hydrophobic filter near the transporter binding site confers selectivity. Previous phylogenetic studies have mainly surveyed the evolutionary relationships between CPAs of limited diversity[18,19,20,21] These analyses divided CPAs into CPA1 and CPA2, which occasionally are stated to relate to the phenotypical electroneutral/ electrogenic partition[6,8,22,23]. The exact molecular determinants that confer ion selectivity are still unclear
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