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 -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 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 -phosphate group of ATP to the Asp is achieved, analogous molecular modeling of the M-M 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 and Ile of the -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 in the N-domain, the other one close to Asp in the P-domain. However, binding of Mg•ATP to any of these sites in the "open conformation" may not lead to phosphorylation of Asp. Additional conformations of the cytoplasmic loop were found wobbling between "open conformation" <==> "semi-open conformation <==> "closed conformation" in the absence of 2Mg•ATP. The cytoplasmic loop's conformational change to the "semi-open conformation"-characterized by a hydrogen bond between Arg and Asp-triggers by binding of 2Mg•ATP to a single ATP site and conversion to the "closed conformation" the phosphorylation of Asp in the P-domain, and hence the start of Na/K-activated ATP hydrolysis.