Flexibility within the rotor and stators of the vacuolar H+-ATPase

Abstract

The V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V1) to a multistroke proton pump (Vo). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V1 domain relative to the Vo domain to a maximum of ~30°. This has been complemented by elastic network normal mode analysis that shows both flexing and twisting with the compliance being located in the rotor axle, stator filaments, or both. This study provides direct evidence of flexibility within the V-ATPase and by implication in related rotary ATPases, a feature predicted to be important for regulation and their high energetic efficiencies.

Figure 1. Subunit organisation in the rotary ATPase family.

In the V-ATPase, the D/F/d/c-ring structure is the rotor, (AB)₃/(EG)₃/C/H/a is the stator. Equivalent subunits within each complex are in the same colour.

Figure 2. Negative stain electron microscopy of the Saccharomyces and Manduca sexta V-ATPases.

(A) A representative class of yeast V-ATPase alongside the 3 masks used to extract the V₁ (i), central (ii) and V_o domains (iii). (B, C) Yeast V-ATPase classes of particles belonging to the same orientation, as determined from multi-reference alignment, re-aligned to V_o and classified using a mask over V₁. (D) Yeast V-ATPase particles of particular views aligned against V₁ and classified by V_o. Numbers in the bottom right corner of B-D, are particle numbers in each class. (E, F) M. sexta V-ATPase classes of particles belonging to the same orientation and aligned against V₁ and classified around V_o. (G) M. sexta V-ATPase class (far left) and representative views of some of the particles making up the class. In all cases scale bars represent 120Å.

Figure 3. Cryo-EM analysis of Flexibility in Manduca V-ATPase.

(A, B) Single particle cryo-EM analysis of the M. sexta V-ATPase enzyme aligned against V₁ and classified against V_o. (C) Reprojections from the M. sexta reconstruction showing the greatest displacement of V₁ relative to V_o. Scale bars represent 100Å.

Figure 4. The influence of ATP on V-ATPase flexibility.

The classes which represented the most degree of flexing between V₁ and V_o for the M-sexta V-ATPase in the absence (A) and presence (B) of 5mM ATP. All data were aligned to the same references and classified in Imagic-5, using the circular mask shown in the top right corner. The Scale bar represents 150Å. The full set of 100 classes from which these were extracted are shown in Figure S2.

Figure 5. Deformation of the yeast V-ATPase along the first three non-trivial normal modes as calculated for the 256-bead ERNM.

(A) Extreme conformers are depicted as a coarse-grained representation and as interpolated density maps, where the two motors are rotated against each other (left) or bended (middle, right). (B) Blue arrows represent the eigenvector corresponding to the first non-zero normal mode, which corresponds to twisting of the whole complex, consistent with the rotary mechanism of the V-ATPase. (C, D) The second and third modes of motion are bending motions with the soluble motor flexing (V₁) with respect to the membrane rotor domain (V_o) either back-forth (C) or side-by-side (D) suggesting that V-ATPase is laterally compliant.

Figure 6. Comparisons between the NMA models and negative stain classes.

(A) Negative stain class of M. sexta V-ATPase showing the maximum flexing between V₁ and V_o relative to each other. A view of the molecular dynamic simulation in the most “flexed” state of M. sexta V-ATPase as a map representation (B) and atom representation (C) shown in the same orientation as (A) with the equivalent flexing of V₁ relative to V_o.