Ken Yokoyama

Mammalian mitochondrial ATP synthase typically organizes into rows of V-shaped dimers that impose significant membrane curvature essential for mitochondrial cristae formation. Using gentle, column-free purification combined with single-particle cryo-electron microscopy, we have identified a previously unrecognized planar dimeric form of bovine ATP synthase exhibiting minimal membrane bending. This planar dimer is characterized structurally by anti-parallel arrangement of two ATP synthase complexes linked by a straight conformation of inhibitory factor 1 (IF1), a sharp contrast to the kinked IF1 observed in tetrameric assemblies. Molecular dynamics simulations confirm that transitioning between straight and kinked IF1 conformations occurs without significant energetic barriers. The planar dimer also displays distinct peripheral stalk positioning relative to its adjacent α subunit. These structural divergences suggest a specialized function and a distinct localization for planar ATP synthase dimers, providing structural support for a division of labor within mitochondrial ATP synthase populations.

Understanding the functional mechanisms of membrane protein complexes requires structural analysis within their native membrane environment. Here, we applied cryo-electron microscopy to determine the structures of F_oF₁ ATP synthase and respiratory supercomplexes (SCs) on sub-mitochondrial particles (SMPs) isolated from bovine heart mitochondria. Most F_oF₁ complexes were observed as dimers stabilized by the regulatory factor IF₁, and a tetrameric assembly comprising two F_oF₁-IF₁ dimers arranged linearly was also identified. This finding indicates that the tetrameric units of F_oF₁ are present in the mitochondrial inner membrane and contribute to shaping cristae tips in mammalian mitochondria. F_o domain maps resolve the e-subunit- c₈-ring interface and show no discrete density for a tightly bound lipid within the c₈-ring. In addition to the previously reported SCs compositions CI₁CIII₂CIV₁ and CI₁CIII₂CIV₂, our analysis identified an additional assembly with the composition CI₁CIII₂CIV₃, as well as a CI₂CIII₂CIV₆ mega-complex. This approach enables rapid structural determination of F_oF₁ ATP synthase and SCs from minimal membrane fractions, providing a foundation for elucidating the molecular basis of metabolic disorders and mitochondrial diseases at the level of higher-order architecture.

ATP synthases are rotary molecular machines that use the proton motive force to rotate the central rotor complex relative to the surrounding stator apparatus, thereby coupling the ATP synthesis. We reconstituted the V/A-ATPase into liposomes and performed structural analysis using cryo-EM under conditions where the proton motive force was applied in the presence of ADP and Pi. ATP molecules were bound at two of the three catalytic sites of V/A-ATPase, confirming that the structure represents a state adopted during ATP synthesis. In this structure, the catalytic site closes upon binding of ADP and Pi through an induced fit mechanism. Multiple structures were obtained where the membrane-embedded rotor ring was in a different position relative to the stator. By comparing these structures, we found that torsion occurs in both the central rotor and the peripheral stator during 31° rotation of rotor ring. These structural snapshots of V/A-ATPase provide crucial insights into the mechanism of rotary catalysis of ATP synthesis.

Mammalian mitochondrial ATP synthase typically organizes into rows of V-shaped dimers that impose significant membrane curvature essential for mitochondrial cristae formation. Using gentle, column-free purification combined with single-particle cryo-electron microscopy, we have identified a previously unrecognized planar dimeric form of bovine ATP synthase exhibiting minimal membrane bending. This planar dimer is characterized structurally by antiparallel arrangement of two ATP synthase complexes linked by a straight conformation of the inhibitory factor 1 (IF1), a sharp contrast to the kinked IF1 observed in tetrameric assemblies. Molecular dynamics simulations confirm that transitioning between straight and kinked IF1 conformations occurs without significant energetic barriers. The planar dimer also displays distinct peripheral stalk positioning relative to its adjacent α subunit. These structural divergences suggest a specialized functional role and localization distinct from the canonical sharply membrane bending ATP synthase oligomers, providing structural support for a model of membrane curvature-driven division of labor within mitochondrial ATP synthase populations.

ATP synthases play a crucial role in energy production by utilizing the proton motive force (pmf) across the membrane to rotate their membrane-embedded rotor c-ring, and thus driving ATP synthesis in the hydrophilic catalytic hexamer. However, the mechanism of how pmf converts into c-ring rotation remains unclear. This study presents a 2.8 Å cryo-EM structure of the V_o domain of V/A-ATPase from Thermus thermophilus, revealing precise orientations of glutamate (Glu) residues in the c₁₂-ring. Three Glu residues face a water channel, with one forming a salt bridge with the Arginine in the stator (a/Arg). Molecular dynamics (MD) simulations show that protonation of specific Glu residues triggers unidirectional Brownian motion of the c₁₂-ring towards ATP synthesis. When the key Glu remains unprotonated, the salt bridge persists, blocking rotation. These findings suggest that asymmetry in the protonation of c/Glu residues biases c₁₂-ring movement, facilitating rotation and ATP synthesis.

F₁ domain of ATP synthase is a rotary ATPase complex in which rotation of central γ-subunit proceeds in 120° steps against a surrounding α₃β₃ fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic αβ dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F₁ domain in F_oF₁ synthase from Bacillus PS3 sp. during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80° rotation occur simultaneously in F₁ domain when nucleotides are bound at all the three catalytic αβ dimers. The remaining 40° rotation of the complete 120° step is driven by completion of ATP hydrolysis at α_Dβ_D, and proceeds through three sub-steps (83°, 91°, 101°, and 120°) with three associated conformational intermediates. All sub-steps except for one between 91° and 101° associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40° rotation is largely driven by release of intramolecular strain accumulated by the 80° rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.

ATP synthase is the most crucial enzyme involved in synthesizing ATP within the cell, and it can also be the cause of mitochondrial diseases due to certain mutations. The authors successfully purified FoF1 from HEK cells and obtained a high-resolution structure using CryoEM structural analysis. The structural information obtained in this study provides valuable data for drug development targeting FoF1.