Abstract
Vigna radiata H+–translocating pyrophosphatases (VrH+-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H+ into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H+-PPases has been elucidated, the H+ translocation mechanism of H+-PPases in the solution state remains unclear. In this study, we used hydrogen–deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H+-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H+-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H+-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston.
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Abbreviations
- DDM:
-
n-Dodecyl-β-d-maltoside
- D2O:
-
Deuterium oxide
- IDP:
-
Imidodiphosphate
- MALDI:
-
Matrix-assisted laser desorption/ionization
- VrH+-PPase:
-
Vigna radiata H+–translocating pyrophosphatase
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L-KH: Formal analysis, Writing - Original Draft Visualization, Writing - Review & Editing Y-CH: Validation Formal analysis, Investigation, Data Curation P-CC: ValidationFormal analysis, Investigation C-HL: Validation S-ML: Validation Y-HHH: Methodology Resources, Data Curation, Writing - Review & Editing, Supervision, Project administration R-LP: Conceptualization Ideas, Resources Writing - Review & Editing, Supervision, Project administration
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Huang, LK., Huang, YC., Chen, PC. et al. Exploration of the Catalytic Cycle Dynamics of Vigna Radiata H+–Translocating Pyrophosphatases Through Hydrogen–Deuterium Exchange Mass Spectrometry. J Membrane Biol 256, 443–458 (2023). https://doi.org/10.1007/s00232-023-00295-9
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DOI: https://doi.org/10.1007/s00232-023-00295-9