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+.
Laboratoire de Pharmacodynamie et de Thérapeutique, Faculté de Médecine, Bât. GE, 808 route de Lennik, B-1070, Brussels, Belgium.
R I
RI
Saba
E
E
Goormaghtigh
J M
JM
Ruysschaert
A
A
Herchuelz
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D006868:Hydrolysis; D015151:Immunoblotting; D008024:Ligands; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D008024:Ligands; D008563:Membrane Lipids; D010455:Peptides; D019831:Sodium-Calcium Exchanger; C066745:exchanger inhibitory peptide; D019286:Endopeptidase K