The ATP-Mg2+ binding site and cytoplasmic domain interactions of Na+,K+-ATPase investigated with Fe2+-catalyzed oxidative cleavage and molecular modeling.
This work utilizes Fe(2+)-catalyzed cleavages and molecular modeling to obtain insight into conformations of cytoplasmic domains and ATP-Mg(2+) binding sites of Na(+),K(+)-ATPase. In E(1) conformations the ATP-Fe(2+) complex mediates specific cleavages at 712VNDS (P domain) and near 440VAGDA (N domain). In E(2)(K), ATP-Fe(2+) mediates cleavages near 212TGES (A domain), near 440VAGDA, and between residues 460-490 (N domain). Cleavages at high ATP-Fe(2+) concentrations do not support suggestions for two ATP sites. A new reagent, fluorescein-DTPA, has been synthesized. The fluorescein-DTPA-Fe(2+) complex mediates cleavages similar to those mediated by ATP-Fe(2+). The data suggest the existence of N to P domain interactions in E(1)Na, with bound ATP-Fe(2+) or fluorescein-DPTA-Fe(2+), A-N, and A-P interactions in E(2)(K), and provide testable constraints for model building. Molecular models based on the Ca(2+)-ATPase structure are consistent with the predictions. Specifically, high-affinity ATP-Mg(2+) binding in E(1) is explained with the N domain tilted ca. 80 degrees toward the P domain, by comparison with well-separated N and P domains in the Ca-ATPase crystal structure. With ATP-Mg(2+) docked, bound Mg(2+) is close to both D710 (in 710DGVNDS) and D443 (in 440VAGDASE). D710 is known to be crucial for Mg(2+) binding. The cleavage and modeling data imply that D443 could also be a candidate for Mg(2+) binding. Comparison of E(1).ATP,Mg(2+) and E(2) models suggests an explanation of the high or low ATP affinities, respectively. We propose a scheme of ATP-Mg(2+) and Mg(2+) binding and N, P, and A domain interactions in the different conformations of the catalytic cycle.
Patchornik
Guy
G
Department of Biological Chemistry and Biological Mass Spectrometry Facility, Weizmann Institute of Science, Rehovot 76100, Israel.
Munson
Keith
K
Goldshleger
Rivka
R
Shainskaya
Alla
A
Sachs
George
G
Karlish
Steven J D
SJ
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D003593:Cytoplasm / Q000201:enzymology*; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D006868:Hydrolysis; D008958:Models, Molecular; D010084:Oxidation-Reduction; D004369:Pentetic Acid / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
D005296:Ferrous Compounds; D010446:Peptide Fragments; D004369:Pentetic Acid; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D019793:Fluorescein