Structure of the Intracellular Ca2+-Binding Domain of NCX1.1 by Site Directed Spin Labeling

The cardiac Na+/Ca2+ exchanger (NCX1.1) serves as the primary means of Ca2+ extrusion from cardiomyocytes following the rise in intracellular Ca2+ during contraction. The exchanger is regulated by binding of Ca2+ to the intracellular domain. This domain is composed of an α-catenin-like domain (CLD) that connects two structurally homologous Ca2+ binding domains (CBD1 and CBD2) to the transmembrane domain of the exchanger. NMR and X-ray crystallographic studies have provided structures for the isolated CBD1 and CBD2 domains and have suggested how Ca2+ binding alters their structures and motional dynamics. It remains unknown how Ca2+ binding to the intact Ca2+ sensor signals the transmembrane domain to regulate exchanger activity. Site directed spin labeling has been employed to address this question. Conventional EPR experiments have shown that residues in the structured β-sandwich regions are insensitive to Ca2+ binding and that the α-helical region of CBD2 remains intact upon Ca2+ binding. Double Electron Electron Resonance (DEER) measurements on doubly labeled constructs revealed that CBD1 and CBD2 are not lengthwise antiparallel in close proximity but rather residues in the distal ends that connect to the CLD are greater than 60 A apart. DEER measurements between inter-domain residues nearer to the apex of the Ca2+ sensor are in close enough proximity to be measured by DEER and these distances are sensitive to Ca2+ binding. These inter-domain distances have been employed to construct a working structural model for CBD12. The current studies are in reasonable agreement with SAXS studies by Hilge et al (PNAS 106:14333-8, 2009) and provide new insight into a structural rearrangement of the intact Ca2+ sensor that may be involved in regulation of Na+/Ca2+ exchange.