NMR structure note: solution structure of Ca2+ binding domain 2B of the third isoform of the Na+/Ca2+ exchanger
The Na+/Ca2+ exchanger (NCX) is an omnipresent plasma membrane protein that catalyzes the removal of intracellular Ca2+ for the uptake of Na+ [see for review (Lytton 2007)]. Mammals express three isoforms, denoted as NCX1, NCX2, or NCX3, of which isoforms 1 and 3 also display alternative splicing. The exchanger is activated by allosteric Ca2+ binding to Ca2+ binding domains 1 and 2 (CBD1 and CBD2) located in the large cytosolic loop between transmembrane helix 5 and 6. CBD1 binds four Ca2+ ions, irrespective of the isoform and is considered the primary Ca2+ sensor. The number of Ca2+ ions that bind to CBD2 depends on the isoform and splice variant. It is hypothesized that the number of binding sites determines the ability of the exchanger to overcome the Na+ dependent inactivation (Hilge et al. 2009) and that Mg2+ binding plays a modulating role (Boyman et al. 2009; Breukels et al. 2011).
NCX1 is thus far the best-studied isoform and solution or X-ray structures of...
KeywordsResonance Assignment Isothermal Titration Calorimetry Backbone Dynamic Alignment Medium Side Chain Chemical Shift
Ca2+ binding domain 2
Macroscopic dissociation constant
Isothermal titration calorimetry
Na+/Ca2+ exchanger isoform 3
Nuclear Overhauser effect
Optical density at 600 nm
Residual dipolar coupling
Root mean square
Root mean square deviation
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
We thank Jurgen Doreleijers for assistance with Xplor-NIH and iCing, Gjalt van Rutten for support with using the HPC Cloud infrastructure at BitBrains. This work was supported by grants from the Netherlands Organization for Scientific Research (NWO): 700.55.443 and 700.57.101.
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