Effect of cations on ATP binding to the N-domain of Na+, K+-ATPase

The nucleotide-binding domain (N-domain) of the Na ⁺ , K ⁺ -ATPase (NKA) is physicochemically characterized by a high content of Glu and Asp residues, resulting in a low isoelectric point (pI = 5.0). Acidic proteins are known to interact with cations. The analysis in silico revealed potential cation interaction sites in the NKA N-domain structure. The interaction with cations was tested in vitro by using a recombinant NKA N-domain. The N-domain contains two Trp residues at the protein surface, as determined by acrylamide-mediated fluorescence quenching, that are useful for structural studies through fluorescence changes. Intrinsic fluorescence of the N-domain was quenched by the presence of cations (Na ⁺ , K ⁺ , Ca ²⁺ ) indicating an effect on the protein structure. ATP binding also quenched the N-domain intrinsic fluorescence, which allowed nucleotide affinity determination and sigmoid kinetics for binding. In the presence of cations, the N-domain affinity for ATP was increased, as well as binding cooperativity. Molecular docking of fluorescein isothiocyanate (FITC) with the N-domain showed two binding modes with the isothiocyanate group located 5–6 Å close to Lys480 and Lys501 in the nucleotide-binding site. The presence of ATP prevented the FITC covalent labeling of the N-domain demonstrating the competitive behavior for the binding site. Finally, it was found that the presence of Na ⁺ and K ⁺ , but not Ca ²⁺ , diminished the FITC labeling of the N-domain; i.e., by decreasing FITC affinity at the nucleotide-binding site. It is proposed that cations interact with the N-domain structure and thereby modulate nucleotide (ATP) affinity and possibly affecting NKA catalysis.