Na<sup>+</sup>,K<sup>+</sup>-ATPase with Disrupted Na<sup>+</sup> Binding Sites I and III Binds Na<sup>+</sup> with Increased Affinity at Site II and Undergoes Na<sup>+</sup>-Activated Phosphorylation with ATP
Na<sup>+</sup>,K<sup>+</sup>-ATPase actively extrudes three cytoplasmic Na<sup>+</sup> ions in exchange for two extracellular K<sup>+</sup> ions for each ATP hydrolyzed. The atomic structure with bound Na<sup>+</sup> identifies three Na<...
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MDPI AG
2024-01-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/14/1/135 |
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| author | Hang N. Nielsen Rikke Holm Ryan Sweazey Jens Peter Andersen Pablo Artigas Bente Vilsen |
| author_facet | Hang N. Nielsen Rikke Holm Ryan Sweazey Jens Peter Andersen Pablo Artigas Bente Vilsen |
| author_sort | Hang N. Nielsen |
| collection | DOAJ |
| description | Na<sup>+</sup>,K<sup>+</sup>-ATPase actively extrudes three cytoplasmic Na<sup>+</sup> ions in exchange for two extracellular K<sup>+</sup> ions for each ATP hydrolyzed. The atomic structure with bound Na<sup>+</sup> identifies three Na<sup>+</sup> sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na<sup>+</sup> binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na<sup>+</sup> sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na<sup>+</sup> sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na<sup>+</sup> access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na<sup>+</sup> affinity. This indicates that Na<sup>+</sup> binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E<sub>1</sub>-like configuration, allowing Na<sup>+</sup> but not K<sup>+</sup> to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector. |
| first_indexed | 2024-03-08T11:04:31Z |
| format | Article |
| id | doaj.art-8d61d63be8a847e59e1d525995cf6e1d |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-08T11:04:31Z |
| publishDate | 2024-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-8d61d63be8a847e59e1d525995cf6e1d2024-01-26T15:22:28ZengMDPI AGBiomolecules2218-273X2024-01-0114113510.3390/biom14010135Na<sup>+</sup>,K<sup>+</sup>-ATPase with Disrupted Na<sup>+</sup> Binding Sites I and III Binds Na<sup>+</sup> with Increased Affinity at Site II and Undergoes Na<sup>+</sup>-Activated Phosphorylation with ATPHang N. Nielsen0Rikke Holm1Ryan Sweazey2Jens Peter Andersen3Pablo Artigas4Bente Vilsen5Department of Biomedicine, Aarhus University, DK-8000 Aarhus, DenmarkDepartment of Biomedicine, Aarhus University, DK-8000 Aarhus, DenmarkDepartment of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USADepartment of Biomedicine, Aarhus University, DK-8000 Aarhus, DenmarkDepartment of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USADepartment of Biomedicine, Aarhus University, DK-8000 Aarhus, DenmarkNa<sup>+</sup>,K<sup>+</sup>-ATPase actively extrudes three cytoplasmic Na<sup>+</sup> ions in exchange for two extracellular K<sup>+</sup> ions for each ATP hydrolyzed. The atomic structure with bound Na<sup>+</sup> identifies three Na<sup>+</sup> sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na<sup>+</sup> binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na<sup>+</sup> sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na<sup>+</sup> sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na<sup>+</sup> access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na<sup>+</sup> affinity. This indicates that Na<sup>+</sup> binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E<sub>1</sub>-like configuration, allowing Na<sup>+</sup> but not K<sup>+</sup> to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.https://www.mdpi.com/2218-273X/14/1/135Na<sup>+</sup>,K<sup>+</sup>-pumpNa<sup>+</sup> siteK<sup>+</sup> siteNa<sup>+</sup> affinityK<sup>+</sup> affinityP-type ATPase |
| spellingShingle | Hang N. Nielsen Rikke Holm Ryan Sweazey Jens Peter Andersen Pablo Artigas Bente Vilsen Na<sup>+</sup>,K<sup>+</sup>-ATPase with Disrupted Na<sup>+</sup> Binding Sites I and III Binds Na<sup>+</sup> with Increased Affinity at Site II and Undergoes Na<sup>+</sup>-Activated Phosphorylation with ATP Biomolecules Na<sup>+</sup>,K<sup>+</sup>-pump Na<sup>+</sup> site K<sup>+</sup> site Na<sup>+</sup> affinity K<sup>+</sup> affinity P-type ATPase |
| title | Na<sup>+</sup>,K<sup>+</sup>-ATPase with Disrupted Na<sup>+</sup> Binding Sites I and III Binds Na<sup>+</sup> with Increased Affinity at Site II and Undergoes Na<sup>+</sup>-Activated Phosphorylation with ATP |
| title_full | Na<sup>+</sup>,K<sup>+</sup>-ATPase with Disrupted Na<sup>+</sup> Binding Sites I and III Binds Na<sup>+</sup> with Increased Affinity at Site II and Undergoes Na<sup>+</sup>-Activated Phosphorylation with ATP |
| title_fullStr | Na<sup>+</sup>,K<sup>+</sup>-ATPase with Disrupted Na<sup>+</sup> Binding Sites I and III Binds Na<sup>+</sup> with Increased Affinity at Site II and Undergoes Na<sup>+</sup>-Activated Phosphorylation with ATP |
| title_full_unstemmed | Na<sup>+</sup>,K<sup>+</sup>-ATPase with Disrupted Na<sup>+</sup> Binding Sites I and III Binds Na<sup>+</sup> with Increased Affinity at Site II and Undergoes Na<sup>+</sup>-Activated Phosphorylation with ATP |
| title_short | Na<sup>+</sup>,K<sup>+</sup>-ATPase with Disrupted Na<sup>+</sup> Binding Sites I and III Binds Na<sup>+</sup> with Increased Affinity at Site II and Undergoes Na<sup>+</sup>-Activated Phosphorylation with ATP |
| title_sort | na sup sup k sup sup atpase with disrupted na sup sup binding sites i and iii binds na sup sup with increased affinity at site ii and undergoes na sup sup activated phosphorylation with atp |
| topic | Na<sup>+</sup>,K<sup>+</sup>-pump Na<sup>+</sup> site K<sup>+</sup> site Na<sup>+</sup> affinity K<sup>+</sup> affinity P-type ATPase |
| url | https://www.mdpi.com/2218-273X/14/1/135 |
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