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 V_o 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.

Figure 1

Sample purification. (a) Size exclusion chromatography of r-EhV-ATPase. (b) 16% SDS-PAGE of r-EhV-ATPase. Nine components consisted of all subunits were identified. Molecular markers are shown on both sides (M). Fraction numbers are labeled as 1–9 on the top. The samples of boxed fractions (1–3) were used for subsequent cryo-EM analysis. The full-length gel is presented.

Figure 2

Image of r-EhV-ATPase by ZPP cryo-EM. (a) ZPP cryo-EM image of ice-embedded r-EhV-ATPase particles. Scale bar: 200 nm. (b) Higher magnification image of r-EhV-ATPase particles. Representative particles are labeled with red circles. Scale bar: 50 nm.

Figure 3

Single-particle analysis of r-EhV-ATPase by ZPP cryo-EM. (a) Representative 2D class averages of r-EhV-ATPase particles. 2D class averages were calculated from ~120,000 particles. Scale bar: 10 nm. (b) Gold-standard Fourier shell correlation (GS-FSC) curve for the final 3D reconstruction of r-EhV-ATPase. Resolution was estimated as 17.3 Å using GS-FSC criteria. (c) Angular distribution of individual images in the final 3D reconstruction, showing unbiased image sampling was achieved.

Figure 4

ZPP cryo-EM map of r-EhV-ATPase at 17.3 Å resolution. (a) The cryo-EM map (right) and representative cross-sections (left): a section of cytoplasmic V₁ domain (top panel), section of transmembrane V_o domain (bottom panel), and section between V₁ and V_o domains (middle panel). Each subunit is labeled as A, B, DF, EG, a, d, and c. The map was separately shown with 90° different orientations. Scale bar: 5 nm. (b) Available atomic models were fitted into the cryo-EM map. A₃B₃DF (V₁) complex from PDBID: 3VR4, EG complexes from PDBID: 3K5B (right side model; T. thermophilus), PDBID: 3V6I (left side model; T. thermophilus), c-subunit from PDBID: 2BL2, and a-subunit from PDBID: 5GAS (T. thermophilus). The d-subunit was created by homology modeling as a template of PDBID: 1R5Z (T. thermophilus d-subunit) using LOOPP server.

Figure 5

Interaction between the a- and c-subunits. (a) Homology model of membrane-associated C-terminal half of the Eh a-subunit (magenta). The original model of the Tt a-subunit (PDBID: 5GAS, light green) was overlaid with the homology model. The conserved arginine is labeled in blue. (b) The calculated maps of the a- and c-subunits were fitted into the cryo-EM map of the transmembrane V_o region. (c) Topological model between Na⁺-bound glutamate in the c-subunit (Glu139, red, PDBID: 2BL2) and conserved arginine (Arg573, blue) in the a-subunit. The closest distance between the a-subunit and c-ring, which is located around Arg573 in a-subunit and Glu139 in c-ring, is indicated. The positions of bound Na⁺ are labeled with purple spheres. A possible entrance for Na⁺ is indicated by a red arrow.

Figure 6

r-EhV-ATPase-Fab shows a different catalytic state. (a) Cryo-EM map of r-EhV-ATPase-Fab fitted by the atomic model of bound Fab fragment (PDBID: 4YO0). Scale bar: 5 nm. (b–d) Sections of V₁, joint, and V_o regions in cryo-EM maps with and without PA tag system, whose positions are indicated in (a), respectively. The r-EhV-ATPase-Fab shows a different state from that without the PA tag system. Red arrowheads show ATP-binding pockets. Red arrows show different orientations of the subunits with and without PA tag system.

Figure 7

Off-axis rotor in EhV-ATPase. (a) In EhV-ATPase, the central rotors and peripheral stalks were tilted from the central axis of the c-ring (red and yellow lines). The geometric centers of the d-subunits were also shifted from the central axis of the c-ring (lower panel). (b) In TtV/A-ATPase, the central rotors and peripheral stalks were nearly parallel to the central axis of the c-ring. The geometric centers of the d-subunits nearly coincided with the center of the c-ring (lower panel). (c) In ScV-ATPase, the central rotors were nearly parallel to the central axis of the c-ring, though the symmetrical three peripheral stalks are tilted from the central axis of the c-ring. The geometric centers of the d-subunits nearly coincided with the center of the c-ring (lower panel).

Figure 8

Interactions between a- and c-subunits. EhV-ATPase (a), TtV/A-ATPase (b), ScV-ATPase (c). The interface sizes (indicated with yellow dotted lines) were estimated from the solvent-accessible surface areas using PyMOL software. Conserved Arg573 and Glu139 are located between the a- and c-subunits (asterisks).