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 Fe2+-catalyzed cleavages and molecular modeling to obtain insight into conformations of cytoplasmic domains and ATP-Mg2+ binding sites of Na+,K+-ATPasc. In E-1 conformations the ATP-Fe2+ Complex mediates specific cleavages at 712VNDS (P domain) and near 440VAGDA (N domain). In E-2(K), ATP-Fe2+ mediates cleavages near 212TGES (A domain), near 440VAGDA, and between residues 460-490 (N domain). Cleavages at high ATP-Fe2+ concentrations do not support suggestions for two ATP sites. A new reagent, fluorescein-DTPA, has been synthesized. The fluorescein-DTPA-Fe2+ Complex mediates cleavages similar to those mediated by ATP-Fe2+. The data suggest the existence of N to P domain interactions in E1Na, with bound ATP-Fe2+ or fluorescein-DPTA-Fe2+, A-N, and A-P interactions in E-2(K), and provide testable constraints for model building. Molecular models based on the Ca2+-ATPase structure are consistent with the predictions. Specifically, high-affinity ATP-Mg2+ binding in E-1 is explained with the N domain tilted ca. 80degrees toward the P domain, by comparison with well-separated N and P domains in the Ca-ATPase crystal structure. With ATP-Mg2+ docked, bound Mg2+ is close to both D710 (in 710DGVNDS) and D443 (in 440VAGDASE). D710 is known to be crucial for Mg2+ binding. The cleavage and modeling data imply that D443 could also be a candidate for Mg2+ binding. Comparison of E-1.ATP,Mg2+ and E-2 models suggests an explanation of the high or low ATP affinities, respectively. We propose a scheme of ATP-Mg2+ and Mg2+ binding and N, P, and A domain interactions in the different conformations of the catalytic cycle.