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

Jun Tsunoda,Kazuyoshi Murata,Ryota Iino,Chihong Song,Hiroshi Ueno,Fabiana Lica Imai,Takeshi Murata,Junichi Takagi

doi:10.1038/s41598-018-33977-9

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.

Highlights

V-ATPase is a rotary enzyme that forms a large membrane protein complex of approximately 800 kDa
We report the first complete structure of EhV-ATPase determined by Zernike phase plate (ZPP) cryo-electron microscopy (cryo-EM) and single-particle analysis, which suggests a mechanism of ion transportation via the transmembrane Vo domain coupled with ATP hydrolysis in the V1 domain
We applied peptide epitope (PA) tag system[19,20] to stabilize the originally heterogeneous structure caused by the ATP hydrolysis states of the V1 part by stopping the rotation of the central rotor with Fab bound to a braking site

Summary

Introduction

V-ATPase is a rotary enzyme that forms a large membrane protein complex of approximately 800 kDa. We report the first complete structure of EhV-ATPase determined by Zernike phase plate (ZPP) cryo-EM and single-particle analysis, which suggests a mechanism of ion transportation via the transmembrane Vo domain coupled with ATP hydrolysis in the V1 domain. The PA tag sequence introduced to the d-subunit which is a part of the central rotor in EhV-ATPase and the bound Fab unexpectedly revealed the different conformation and identified one minor catalytic conformation. These catalytic conformations with and without Fab coincided with those of the minor state 2 and the major state 1 in TtV/A-ATPase, respectively. The most prominent feature was the off-axis rotor in EhV-ATPase, where the cytoplasmic V1 motor and transmembrane Vo pump were connected to the off-axis rotor subunits

Methods

Results

Conclusion