Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance
- Kyoto Sangyo University, Japan
- Osaka University, Japan
- Tokyo University of Pharmacy and Life Sciences, Japan
Abstract
V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 domain, with proton flow through the Vo membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo domain is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo domain adopts the auto-inhibited form and how the holo complex prevents formation of the inhibited Vo form.
Data availability
The density maps and the built models for Tth VoV1, Tth V1 (focused refined), and Tth Vo were deposited in EMDB (EMDB ID; 30013, 30014, and 30015) and PDB (PDB ID; 6LY8 for V1 and 6LY9 for isolated Vo), respectively. All data is available in the main text or the supplementary materials.
Article and author information
Author details
Funding
Japan Society for the Promotion of Science (17H03648)
- Ken Yokoyama
Japan Agency for Medical Research and Development (JP17am0101001)
- Kaoru Mitsuoka
Ministry of Education, Culture, Sports, Science, and Technology (12024046)
- Kaoru Mitsuoka
Takeda Science Foundation
- Ken Yokoyama
Japan Science and Technology Agency (JPMJCR1865)
- Kaoru Mitsuoka
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Kishikawa et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance
eLife 9:e56862.
https://doi.org/10.7554/eLife.56862
Further reading
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We present near-atomic-resolution cryoEM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9 Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2–2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.
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