The second Ca2+-binding domain of the Na+-Ca2+ exchanger is essential for regulation:: Crystal structures and mutational analysis

The Na+-Ca2+ exchanger plays a central role in cardiac contractility by maintaining Ca2+ homeostasis. Two Ca2+-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca2+ binding to these regulatory domains activates the transport of Ca2+ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca2+ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca2+-bound (1.7-angstrom resolution) and -free (1.4-angstrom resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca2+ ions in primary and secondary Ca2+ sites. In the absence of Ca2+, LyS(585) stabilizes the structure by coordinating two acidic residues (Asp(552) and Glu(648)), one from each of the Ca2+-binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca2+ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca2+ site completely remove Ca2+ regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na+-Ca2+ exchanger.