The second Ca2+-binding domain of the Na+-Ca2+ exchanger is essential for regulation: Crystal structures and mutational analysis
Abstract
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-å resolution) and -free (1.4-å 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+, Lys585 stabilizes the structure by coordinating two acidic residues (Asp552 and Glu648), 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.
- Publication:
-
Proceedings of the National Academy of Science
- Pub Date:
- November 2007
- DOI:
- 10.1073/pnas.0707417104
- Bibcode:
- 2007PNAS..10418467B