Binding of calcium is sensed structurally and dynamically throughout the second calcium-binding domain of the sodium/calcium exchanger.
We report the effects of Ca(2+) binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. Analysis of the Ca(2+)-induced chemical shifts perturbations yields similar K(D) values of 16-24 microM for the two CBD2-AD Ca(2+)-binding sites, and significant effects are observed up to 20 A away. To quantify the Ca(2+)-induced chemical shift changes, we performed a comparative analysis of eight Ca(2+)-binding proteins that revealed large differences between different protein folds. The CBD2 (15)N relaxation data show the CBD2-AD Ca(2+) coordinating loops to be more rigid in the Ca(2+)-bound state as well as to affect the FG-loop located at the opposite site of the domain. The equivalent loops of the CBD2-BD splice variant do not bind Ca(2+) and are much more dynamic relative to both the Ca(2+)-bound and apo forms of CBD2-AD. A more structured FG-loop in CBD2-BD is suggested by increased S(2) order parameter values relative to both forms of CBD2-AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca(2+)-binding event is felt throughout the molecule. The data suggest that the FG-loop plays an important role in connecting the Ca(2+)-binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG-loop with the first Ca(2+)-binding domain of NCX.
Breukels
Vincent
V
Protein Biophysics, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands.
Vuister
Geerten W
GW
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004285:Dogs; D009682:Magnetic Resonance Spectroscopy; D009038:Motion; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000378:metabolism*; D017186:Titrimetry
D019831:Sodium-Calcium Exchanger; D002118:Calcium