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⁺-ATPase. In E₁ conformations the ATP-Fe²⁺ complex mediates specific cleavages at 712VNDS (P domain) and near 440VAGDA (N domain). In E₂(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 E₂(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 E₁ is explained with the N domain tilted ca. 80° 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 E₁·ATP,Mg²⁺ and E₂ 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.