Off-axis rotor in Enterococcus hirae V-ATPase visualized by Zernike phase plate single-particle cryo-electron microscopy

Abstract

EhV-ATPase is an ATP-driven Na+ pump in the eubacteria Enterococcus hirae (Eh). Here, we present the first entire structure of detergent-solubilized EhV-ATPase by single-particle cryo-electron microscopy (cryo-EM) using Zernike phase plate. The cryo-EM map dominantly showed one of three catalytic conformations in this rotary enzyme. To further stabilize the originally heterogeneous structure caused by the ATP hydrolysis states of the V1-ATPases, a peptide epitope tag system was adopted, in which the inserted peptide epitope sequence interfered with rotation of the central rotor by binding the Fab. As a result, the map unexpectedly showed another catalytic conformation of EhV-ATPase. Interestingly, these two conformations identified with and without Fab conversely coincided with those of the minor state 2 and the major state 1 of Thermus thermophilus V/A-ATPase, respectively. The most prominent feature in EhV-ATPase was the off-axis rotor, where the cytoplasmic V1 domain was connected to the transmembrane Vo domain through the off-axis central rotor. Furthermore, compared to the structure of ATP synthases, the larger size of the interface between the transmembrane a-subunit and c-ring of EhV-ATPase would be more advantageous for active ion pumping.

Single-molecule analysis reveals rotational substeps and chemo-mechanical coupling scheme of Enterococcus hirae V1-ATPase

“…The V-ATPase is composed of two rotary motors, a membrane-embedded V o transporting ions and a water-soluble V 1 -ATPase (V 1 ) hydrolyzing ATP, which are connected by a central stalk and either two (EhV-ATPase and V/A-ATPase) or three (eukaryotic V-ATPase) peripheral stalks (8–13). The V o is composed of the channel-forming a subunit, the ion binding ring of the c subunits, and the d subunit and the EG subcomplex, which form the central and peripheral stalks, respectively (14, 15).…”

Advances in domain and subunit localization technology for electron microscopy

“…Such a range of tools mean that, for a particular experimental situation, the appropriate properties of the labeling approach including, the type of modification, its size, its affinity and the nature of the chemisty used for carrying out the labeling reaction can all be optimized. One such tool, the PA tag/NZ-1 antibody system has been employed for a wide range of uses such as protein purification (Fujii et al 2014), Western blotting, cell flow cytometry (Fujii et al 2014), conformational reporting (Fujii et al 2016), domain level tagging (Wang et al 2018a, b;, and to influence conformational states of target biomolecules (Tsunoda et al 2018). We hope that this review has alerted the biophysics community to this toolbox of intermediate resolution level approaches, which can provide extra structural information in EM experimentation when full atomic detail is not attainable for experimental reasons.…”

“…The type II β-turn conformation of the peptide allows the insertion of the peptide into central domains to give a mobile EM labeling system. Such a system has other uses, for example if the PA peptide is carefully inserted into a rotary protein such as the V-ATPase, then binding of NZ-1 can be used to lock the complex into a particular conformation (Tsunoda et al 2018). By locking the conformational state of a protein, previously rare structural states can be increased, allowing their structural determination using cryo-EM.…”

Biophysical research in Okazaki, Japan

“…Kazuyoshi Murata operates a series of electron microscopes (EM) complementarily to visualize medical and biological targets that span the molecular to cellular scales. While a high-voltage EM (H-1250 M: 1 MV) is designed for observing microorganisms and cells, a phase-contrast cryo-EM (JEM2200FS: 200 kV) is equipped with a Zernike phase plate to achieve high-resolution structural analyses of membrane proteins (Tsunoda et al 2018) and viruses (Okamoto et al 2018). These advanced EMs and analytical techniques are shared with research communities, including the biophysics community, through joint research projects.…”