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
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
15222743
DOI
10.1021/bi0496485
Knihovny.cz E-resources
- MeSH
- Adenosine chemistry MeSH
- Adenosine Triphosphate analogs & derivatives chemistry MeSH
- Amino Acids chemistry MeSH
- Arginine chemistry MeSH
- Point Mutation MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Fluorescent Dyes chemistry MeSH
- Genetic Vectors MeSH
- Glutathione Transferase metabolism MeSH
- Hydrolysis MeSH
- Kinetics MeSH
- Crystallography, X-Ray MeSH
- Aspartic Acid chemistry MeSH
- Glutamic Acid chemistry MeSH
- Ligands MeSH
- Magnetic Resonance Spectroscopy MeSH
- Models, Molecular MeSH
- Molecular Sequence Data MeSH
- Mutation MeSH
- Mice MeSH
- Nucleotides chemistry MeSH
- Proline chemistry MeSH
- Recombinant Fusion Proteins chemistry MeSH
- Amino Acid Sequence MeSH
- Sequence Homology, Amino Acid MeSH
- Serine chemistry MeSH
- Sodium-Potassium-Exchanging ATPase chemistry MeSH
- Software MeSH
- Spectrophotometry MeSH
- Temperature MeSH
- Protein Structure, Tertiary MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Hydrogen chemistry MeSH
- Hydrogen Bonding MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- 2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate MeSH Browser
- Adenosine MeSH
- Adenosine Triphosphate MeSH
- Amino Acids MeSH
- Arginine MeSH
- Fluorescent Dyes MeSH
- Glutathione Transferase MeSH
- Aspartic Acid MeSH
- Glutamic Acid MeSH
- Ligands MeSH
- Nucleotides MeSH
- Proline MeSH
- Recombinant Fusion Proteins MeSH
- Serine MeSH
- Sodium-Potassium-Exchanging ATPase MeSH
- Hydrogen MeSH
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.
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