Cryo-EM and MD infer water-mediated proton transport and autoinhibition mechanisms of Vo complex

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Science Advances  07 Oct 2020:
Vol. 6, no. 41, eabb9605
DOI: 10.1126/sciadv.abb9605
  • Fig. 1 A 2.7-Å cryo-EM structure of yeast Vo in lipid nanodisc.

    (A) A 2.7-Å map of VoND showing subunits a (green), d (cyan), c8 (pink), c′ (orange), c″ (yellow), e (blue), f (purple), and Voa1p (red). The densities for two of the c subunits are removed to allow a view inside the c-ring. Inset, aCT TM α helices 7 and 8 fitted to the cryo-EM density. The annotations of the amino acids follow the traditional color codes of their types. (B) Cross section of the Vo model as indicated by the dashed line in (A). The cytosolic and luminal half-channels are indicated by gray density. The essential glutamic acids of the c-ring are highlighted in spacefill representation. The densities for E137 of c(1) and c(2), E108 of c″, and E145 of c′ are shown in the insets. Distances consistent with hydrogen bond formation to nearby residues are indicated by dashed lines. (C) The central cavity of the c-ring is occupied by lipid molecules (modeled as phosphatidylethanolamine) that are forming a bilayer. (D) Surface representation of aCT as seen from the c-ring showing two cavities (next to the cytosolic and luminal half-channels) that are occupied by nonproteinaceous densities. The cavity on the cytosolic side is occupied by a tightly bound phospholipid (left). The cavity open to the luminal side is occupied by density that we modeled as the glycosylation precursor dolichol-P-P-(GlcNAc)2Manx (right). The pyrophosphate moiety is stabilized by the side chains of aK538, aK593, aS534, and the backbone amide of aL608.

  • Fig. 2 MSPs stabilize nanodisc reconstituted Vo proton channel.

    (A) Focused classification resolves three distinct belts of density that surround the membrane-exposed surface of the c-ring. The three belts were modeled as one canonical dimer and one monomer of MSP. (B) The three belts show varying distances to the c-ring. Whereas the middle belt leaves sufficient space to accommodate a layer of lipid molecules, the upper and lower belts appear to directly contact and stabilize the c-ring, presumably via hydrophobic residues of MSP that are oriented toward the c subunits.

  • Fig. 3 Water molecules at the aCT:c-ring interface.

    (A) Cryo-EM density at the aCT:c-ring interface that is modeled as ordered water molecules. Water molecules are represented as red spheres with surrounding density contoured at 0.025 (2.5 root mean square deviation). (B) Left: Zoomed-in view of the interface with modeled water molecules and their Q-scores. Right: Histogram of Q-scores for modeled water molecules. (C) Overlap of the experimentally assigned water molecules (red beads) with computationally derived water occupancy across a 0.5-μs MD simulation (blue dots). (D) Water dynamics was simulated in the pre–H+-transfer state [c(1)E137 protonated, aE789 deprotonated; left image] and post–H+-transfer state [c(1)E137 deprotonated, aE789 protonated; right image]. (E) Occurrence of water wires with 5000 frames accumulated over 0.5-μs MD simulations indicated by blue vertical lines for the pre–H+-transfer state. No water wires were observed for the post–H+-transfer state as indicated by the red line. (F) Two views of a representative coordinate frame with water-wire connecting c(1)E137 and aE789 in the pre–H+-transfer state. The left image is seen parallel to the membrane toward the aCT:c-ring interface. The right image is seen perpendicular to the membrane along the aCT:c-ring interface. Hydrogen and oxygen atoms are shown in white and red, respectively. (G) Representative coordinate frame with c(1)E137 and aE789 seen in the post–H+-transfer state. The two different views are as in (F). The protonated aE789 blocks access for water molecules from the luminal channel. A few water molecules are retained near the ionized c(1)E137.

  • Fig. 4 Two rotary states of autoinhibited yeast Vo.

    About 95% of all particles in the final cryo-EM dataset of VoND, including the particles that constitute the 2.7-Å structure (A), represent rotary state 3. (B) 3D classification of the final dataset revealed a minor population of particles (~5%) that represent a state different from the hitherto described autoinhibited state (state 3) of the complex. In this minor state, which is designated state 3′, the c-ring is rotated clockwise in the direction of proton pumping by about 14°. This rotation of the c-ring brings E137 of c(1) close to aCT’s two essential arginine residues. (C and D) Whereas the d:aNT interaction at the distal domain of aNT seen in the predominant state 3 is preserved in the minor state 3′ (pink dashed circles), the interaction at aNT’s proximal domain is not (red dashed circles). (E) Free energy profile of c-ring rotation for wild type (c″E108; left) and mutant (c″E108A; right). c-ring rotation was simulated using a biasing potential within a solvated membrane to induce a clockwise motion of the ring. (F) Wild-type coordinate files were extracted from the simulation near the energy minima designated as substates A to E in the left panel of (E). Substate TS represents the high-energy transition between substates C and D. Some of the polar residues in the aCT:c-ring interface are highlighted by stick representation. Distances consistent with hydrogen bond formation are indicated by the blue dashed lines.

Supplementary Materials

  • Supplementary Materials

    Cryo-EM and MD infer water-mediated proton transport and autoinhibition mechanisms of Vo complex

    Soung-Hun Roh, Mrinal Shekhar, Grigore Pintilie, Christophe Chipot, Stephan Wilkens, Abhishek Singharoy, Wah Chiu

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