The Hydrogen Bonds between Arg423 and Glu472 and Other Key Residues, Asp443, Ser477, and Pro489, Are Responsible for the Formation and a Different Positioning of TNP-ATP and ATP within the Nucleotide-Binding Site of Na+/K+-ATPase

Mutation of Arg423 at the N-domain of Na+/K+-ATPase resulted in a large decrease of both TNP-ATP and ATP binding. Thus, this residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site. In addition, mutation of Glu472 also caused a large decrease of both TNP-ATP and ATP binding. On the basis of our computer model, we hypothesized that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. To verify this hypothesis, we have also prepared the construct containing both these mutations. Binding of neither TNP-ATP nor ATP to this double mutant differed from binding to any of the single mutants. This strongly supported the existence of the hydrogen bond between Arg423 and Glu472. Similarly, the conserved residue Pro489 seems to be substantial for the proper interaction of the third and fourth β-strands of the N-domain, which both contain residues that take part in ATP binding. Mutation of Asp443 affected only ATP, but not TNP-ATP, binding, suggesting that these ligands adopt different positions in the nucleotide-binding pocket. On the basis of a recently published crystal structure [Håkansson, K. O. (2003) J. Mol. Biol. 332, 1175−1182], we improved our model and computed the interaction of these two ligands with the N-domain. This model is in good agreement with all previously reported spectroscopic data and revealed that Asp443 forms a hydrogen bond with the NH2 group of the adenosine moiety of ATP, but not TNP-ATP.