Computer modelling reveals new conformers of the ATP binding loop of Na(+)/K(+)-ATPase involved in the transphosphorylation process of the sodium pump

Hydrolysis of ATP by Na(+)/K(+)-ATPase, a P-Type ATPase, catalyzing active Na(+) and K(+) transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp(369) to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp(369) is achieved, analogous molecular modeling of the M(4)–M(5) loop of ATPase was performed using the crystal data of Na(+)/K(+)-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr(338) and Ile(760) of the α(2)-subunit of Na(+)/K(+)-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe(475) in the N-domain, the other one close to Asp(369) in the P-domain. However, binding of Mg(2+)•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp(369). Additional conformations of the cytoplasmic loop were found wobbling between “open conformation” <==> “semi-open conformation <==> “closed conformation” in the absence of 2Mg(2+)•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg(543) and Asp(611)—triggers by binding of 2Mg(2+)•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp(369) in the P-domain, and hence the start of Na(+)/K(+)-activated ATP hydrolysis.