D443 of the N domain of Na+,K+-ATPase interacts with the ATP-Mg2+ complex, possibly via a second Mg2+ ion

This paper provides evidence for an interaction of D443 in the N domain of Na+, K+-ATPase with a Mg2+ ion. Wild-type, D443N/A/C and S445A mutants of porcine Na+, K+-ATPase (alpha 1 beta 1) have been expressed in Pichia pastoris. By comparison with wild-type, D443N reduces the turn -over rate by about 40%. Binding affinity of ATP, measured directly, was not affected by D443N, D443A, or D443C mutations. AMP-PNP-Fe2+-catalyzed oxidative cleavage of Na+,K+-ATPase produces two characteristic fragments, at (VNDS)-V-708 (P domain) and near (440)VAGDA (N domain), respectively. In the D443N and D443A mutants, both cleavages are suppressed, indicating an interaction between the residues with AMPPNP-Fe2+ bound. Previous work suggested that with ATP-Fe2+ bound the N and P domains come into proximity, both D710 and D443 making contact with a single Fe2+= (or Mg2+) ion. However, the crystal structure of Ca2+-ATPase with bound AMP-PCP and Mg2+ confirm the involvement of D703 (D710) but show that E439 (D443) is too far to make contact with the Mg2+. By contrast, in the crystal structure with bound ADP, AlF4, and Mg2+, representing the E-1-P conformation, two Mg2+ ions were observed. Significantly, ADP-Fe2+-mediated oxidative cleavage of renal Na,K-ATPase produces the fragment near (440)VAGDA (N domain), while the cleavage at 708VNDS (P domain) is almost completely absent. The results are explained economically by the hypothesis that ATP is bound with two Mg2+ (Fe2+) ions, a "catalytic" Mg2+ interacting with D710 via the gamma phosphate and a "structural" Mg2+ interacting with D443 via the alpha, and beta phosphates and a water molecule, respectively.