The Intracellular Loop of the Na⁺/Ca²⁺ Exchanger Contains an "Awareness Ribbon"-Shaped Two-Helix Bundle Domain

The Na⁺/Ca²⁺ exchanger (NCX) is a ubiquitous single-chain membrane protein that plays a major role in regulating the intracellular Ca²⁺ homeostasis by the counter transport of Na⁺ and Ca²⁺ across the cell membrane. Other than its prokaryotic counterpart, which contains only the transmembrane domain and is self-sufficient as an active ion transporter, the eukaryotic NCX protein possesses in addition a large intracellular loop that senses intracellular calcium signals and controls the activation of ion transport across the membrane. This provides a necessary layer of regulation for the more complex function of eukaryotic cells. The Ca²⁺ sensor in the intracellular loop is known as the Ca²⁺-binding domain (CBD12). However, how the signaling of the allosteric intracellular Ca²⁺ binding propagates and results in transmembrane ion transportation still lacks a detailed explanation. Further structural and dynamics characterization of the intracellular loop flanking both sides of CBD12 is therefore imperative. Here, we report the identification and characterization of another structured domain that is N-terminal to CBD12 in the intracellular loop using solution nuclear magnetic resonance (NMR) spectroscopy. The atomistic structure of this domain reveals that two tandem long α-helices, connected by a short linker, form a stable crossover two-helix bundle (THB), resembling an "awareness ribbon". Considering the highly conserved amino acid sequence of the THB domain, the detailed structural and dynamics properties of the THB domain will be common among NCXs from different species and will contribute toward the understanding of the regulatory mechanism of eukaryotic Na⁺/Ca²⁺ exchangers.