Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump
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Abstract
Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, 3-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information how the transfer of the γ‑phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4-M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of 2 ATP binding sites in the open conformation, the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation” <==> “semi-open conformation <==> “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation” -- characterized by a hydrogen bond between Arg543 and Asp611 -- triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.
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2017. Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump. PeerJ Preprints 5:e2812v1 https://doi.org/10.7287/peerj.preprints.2812v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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Supplemental Information
Figure S1
The multialignment of the chosen target (P50993, AT1A2_HUMAN) sequence and the two templates (3B8E, 3KDP) for open conformation was prepared by MODELLER program (salign module).
Figure S2
The multialignment of the chosen target (P50993, AT1A2_HUMAN) sequence and the three templates (3WGU, 3WGV, 4HQJ) for closed conformation was prepared by MODELLER program (salign module).
Figure S3
The Berendsen coupling method ( Berendsen, Postma et al. 1984 ) was employed for the temperature and pressure coupling of a system to reflect the reference temperature of 300K and the pressure of 1bar. The leap-frog integration with 104 steps was used for stabilization, with integration step of 1fs, corresponding to 10ps simulation time to reach the equilibrium of the rectangular box. This stabilized rectangular box was used for the main thirty simulations with 5x106 steps (2 fs single step), corresponding to 10ns for each stabilization using the same simulation parameters as for the box stabilization.
Figure S4
The Berendsen coupling method ( Berendsen, Postma et al. 1984 ) was employed for the temperature and pressure coupling of a system to reflect the reference temperature of 300K and the pressure of 1bar. The leap-frog integration with 104 steps was used for stabilization, with integration step of 1fs, corresponding to 10ps simulation time to reach the equilibrium of the rectangular box. This stabilized rectangular box was used for the main thirty simulations with 5x106 steps (2 fs single step), corresponding to 10ns for each stabilization using the same simulation parameters as for the box stabilization.
Figure S5
The Berendsen coupling method ( Berendsen, Postma et al. 1984 ) was employed for the temperature and pressure coupling of a system to reflect the reference temperature of 300K and the pressure of 1bar. The leap-frog integration with 104 steps was used for stabilization, with integration step of 1fs, corresponding to 10ps simulation time to reach the equilibrium of the rectangular box. This stabilized rectangular box was used for the main thirty simulations with 5x106 steps (2 fs single step), corresponding to 10ns for each stabilization using the same simulation parameters as for the box stabilization.
Figure S6
The Berendsen coupling method ( Berendsen, Postma et al. 1984 ) was employed for the temperature and pressure coupling of a system to reflect the reference temperature of 300K and the pressure of 1bar. The leap-frog integration with 104 steps was used for stabilization, with integration step of 1fs, corresponding to 10ps simulation time to reach the equilibrium of the rectangular box. This stabilized rectangular box was used for the main thirty simulations with 5x106 steps (2 fs single step), corresponding to 10ns for each stabilization using the same simulation parameters as for the box stabilization.
Additional Information
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Gracian Tejral conceived and designed the experiments, performed the experiments, wrote the paper, prepared figures and/or tables.
Bruno Sopko conceived and designed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables.
Alois Necas wrote the paper, reviewed drafts of the paper.
Wilhelm Schoner conceived and designed the experiments, wrote the paper, reviewed drafts of the paper.
Evzen Amler conceived and designed the experiments, wrote the paper, reviewed drafts of the paper.
Data Deposition
The following information was supplied regarding data availability:
Comparative modeling of the different conformations are detailed in the Methods section.
Funding
Computational resources were provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085, provided under the programme "Projects of Large Research, Development, and Innovations Infrastructures". This research was supported by the Czech Science Foundation Grant No. 15-15697S, the University Centre for Energy Efficient Buildings (UCEEB) support IPv6; the Ministry of Education, Youth, and Sports of the Czech Republic (National Sustainability Programme I, project No. LO1605; Research Programs NPU I:LO1508 and NPU I:LO1309); the Internal Grant Agency of the Ministry of Health of the Czech Republic (grant No. NT12156 and MZ-VES project no. 16-29680A and 16-28637A); University Hospital Motol (project 9775); and the Ministry of Interior of the Czech Republic (program BV III/1-VS, No VI20152018010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
