Mutation of Arg(423) 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 Glu(472) 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 Arg(423) and Glu(472) 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 Arg(423) and Glu(472). Similarly, the conserved residue Pro(489) seems to be substantial for the proper interaction of the third and fourth beta-strands of the N-domain, which both contain residues that take part in ATP binding. Mutation of Asp(443) 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 Asp(443) forms a hydrogen bond with the NH(2) group of the adenosine moiety of ATP, but not TNP-ATP.

Institute of Physiology Czech Academy of Sciences Vídenská 1083 14220 Prague Czech Republic

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