Conformational changes of the 120-kDa Na+/Ca2+ exchanger protein upon ligand binding: a Fourier transform infrared spectroscopy study.

The 120-kDa Na+/Ca2+ exchanger was purified and reconstituted into lipid vesicles. The secondary structure composition of the exchanger was 39% alpha-helices, 20% beta-sheets, 25% beta-turns, and 16% random coils, as analyzed by Fourier transform infrared attenuated total reflection spectroscopy. The secondary structure composition of the COOH-terminal portion of the protein was compatible with a topology model containing 4-6 transmembrane segments. Furthermore, the secondary structure of the NH2-terminal portion of the cytoplasmic loop was analyzed and found to be different from that of the COOH-terminal portion. Ca2+ and/or the exchange inhibitory peptide (XIP) failed to affect the secondary structure of the 120-kDa protein. Tertiary structure modifications induced by Ca2+ and XIP were analyzed by monitoring the hydrogen/deuterium exchange rate for the reconstituted exchanger. In the absence of ligand, 51% of the protein was accessible to solvent. Ca2+ decreased accessibility to 40%, implicating the shielding of at least 103 amino acids. When both Ca2+ and XIP were added, accessibility increased to 66%. No modification was obtained when XIP was added alone. Likewise, in the presence of Ca2+, XIP failed to modify the tertiary structure of the 70-kDa protein, suggesting that XIP acts at the level of the COOH-terminal portion of the intracellular loop. The present data describe, for the first time, conformational changes of the Na+/Ca2+ exchanger induced by Ca2+ and XIP, compatible with an interaction model where regulatory Ca2+ and inhibitory XIP bind to distinct sites, and where XIP binding requires the presence of Ca2+.