Structure of mammalian V-ATPase with the TLDc domain protein mEAK7 bound

V-ATPases are rotary proton pumps that serve as signaling hubs, with numerous proposed binding partners in cells. We used cryoEM to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~1.6% of complexes bound mEAK7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK7 shows that mEAK7's TLDc domain, which is found in other proteins proposed to bind V-ATPase, interacts with V-ATPase's stator while its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, exogenous mEAK7 does not inhibit purified V-ATPase activity and mEAK7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that interaction of mEAK7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Together, these results reveal how TLDc domains bind V-ATPases and suggest that V-ATPase binding proteins can form labile interactions that are sensitive to the enzyme's activity.

The six steps of the complete F1-ATPase rotary catalytic cycle

F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalysis mechanism. Isolated F1-ATPase catalytic cores can hydrolyze ATP, passing through six intermediate conformational states to generate rotation of their central γ-subunit. Although previous structural studies have contributed greatly to understanding rotary catalysis in the F1-ATPase, the structure of an important conformational state (the binding-dwell) has remained elusive. Here, we exploit temperature and time-resolved cryo-electron microscopy to determine the structure of the binding- and catalytic-dwell states of Bacillus PS3 F1-ATPase. Each state shows three catalytic β-subunits in different conformations, establishing the complete set of six states taken up during the catalytic cycle and providing molecular details for both the ATP binding and hydrolysis strokes. We also identify a potential phosphate-release tunnel that indicates how ADP and phosphate binding are coordinated during synthesis. Overall these findings provide a structural basis for the entire F1-ATPase catalytic cycle.

The six steps of the F1-ATPase rotary catalytic cycle

F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalysis mechanism. When isolated, its F1-ATPase catalytic core can hydrolyze ATP, rotating its γ rotor subunit. Although previous structural studies have contributed greatly to understanding rotary catalysis in F1, the structure of one major conformational state detected in single-molecule studies, termed the binding dwell state, has not yet been determined. Here, by exploiting a temperature-sensitive F1-ATPase mutant from Bacillus PS3, the structure of this binding dwell state was established together with that of the catalytic dwell state. Each state showed three catalytic β subunits in different conformations, providing the complete set of six β subunit conformational states taken up during catalysis cycle. These structures provide molecular details for the power-stroke conformational change that occurs upon ATP binding and induces a ~80° γ subunit rotation, as well as a second torque-generating conformational change, triggered by hydrolysis and product release, that produces a ~40° rotation. This study also identifies a putative phosphate-releasing tunnel that indicates how ADP and phosphate releasing steps are coordinated. Overall these findings provide a structural basis for the entire F1-ATPase rotary catalysis cycle.

Rotary catalysis of bovine mitochondrial F1-ATPase studied by single-molecule experiments

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant βE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted “stall-and-release” experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.

Structure and conformational plasticity of the intact Thermus thermophilus V/A-type ATPase

V (vacuolar)/A (archaeal)-type adenosine triphosphatases (ATPases), found in archaea and eubacteria, couple ATP hydrolysis or synthesis to proton translocation across the plasma membrane using the rotary-catalysis mechanism. They belong to the V-type ATPase family, which differs from the mitochondrial/chloroplast F-type ATP synthases in overall architecture. We solved cryo–electron microscopy structures of the intact Thermus thermophilus V/A-ATPase, reconstituted into lipid nanodiscs, in three rotational states and two substates. These structures indicate substantial flexibility between V1 and Vo in a working enzyme, which results from mechanical competition between central shaft rotation and resistance from the peripheral stalks. We also describe details of adenosine diphosphate inhibition release, V1-Vo torque transmission, and proton translocation, which are relevant for the entire V-type ATPase family.

Structure and Mechanisms of F-Type ATP Synthases

F1Fo ATP synthases produce most of the ATP in the cell. F-type ATP synthases have been investigated for more than 50 years, but a full understanding of their molecular mechanisms has become possible only with the recent structures of complete, functionally competent complexes determined by electron cryo-microscopy (cryo-EM). High-resolution cryo-EM structures offer a wealth of unexpected new insights. The catalytic F1 head rotates with the central γ-subunit for the first part of each ATP-generating power stroke. Joint rotation is enabled by subunit δ/OSCP acting as a flexible hinge between F1 and the peripheral stalk. Subunit a conducts protons to and from the c-ring rotor through two conserved aqueous channels. The channels are separated by ∼6 Å in the hydrophobic core of Fo, resulting in a strong local field that generates torque to drive rotary catalysis in F1. The structure of the chloroplast F1Fo complex explains how ATPase activity is turned off at night by a redox switch. Structures of mitochondrial ATP synthase dimers indicate how they shape the inner membrane cristae. The new cryo-EM structures complete our picture of the ATP synthases and reveal the unique mechanism by which they transform an electrochemical membrane potential into biologically useful chemical energy.

Structural basis of proton translocation and force generation in mitochondrial ATP synthase

ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact mitochondrial ATP synthase dimer by electron cryo-microscopy at near-atomic resolution. Charged and polar residues of the a-subunit stator define two aqueous channels, each spanning one half of the membrane. Passing through a conserved membrane-intrinsic helix hairpin, the lumenal channel protonates an acidic glutamate in the c-ring rotor. Upon ring rotation, the protonated glutamate encounters the matrix channel and deprotonates. An arginine between the two channels prevents proton leakage. The steep potential gradient over the sub-nm inter-channel distance exerts a force on the deprotonated glutamate, resulting in net directional rotation.

Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.