High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane

INTRODUCTION The mitochondrial adenosine triphosphate (ATP) synthase is the enzyme responsible for the synthesis of more than 90% of the ATP produced by mammalian cells under aerobic conditions. The chemiosmotic mechanism, proposed by Peter Mitchell, states that the enzyme transduces the energy of a proton gradient, generated by the electron transport chain, into the major energy currency of the cell, ATP. The enzyme is a large (about 600,000 Da, in the monomer state) multisubunit complex, with a water soluble complex (F 1 ) that contains three active sites and a membrane complex (F o ) that contains the proton translocation pathway, principally comprised of the a subunit and a ring of 10 c subunits, the c 10 -ring (10 in yeast, 8 in mammals). F 1 has a central rotor that, at one end, is within the core of F 1 and, at the other end, is connected to the c 10 -ring of F o . During ATP synthesis, the c 10 -ring rotates, driven by the movement of protons from the cytosol to the mitochondrion, and in turn, the rotor rotates within F 1 in steps of 120 o . The rotation of the rotor causes conformational changes in the catalytic sites, which provides the energy for the phosphorylation of adenosine diphosphate (ADP), as first proposed in the binding-change hypothesis by Paul Boyer. The peripheral stalk acts as a stator connecting F 1 with F o and prevents the futile rotation of F 1 as the rotor spins within it. RATIONALE Structural studies of the ATP synthase have made steady progress since the structure of the F 1 complex was described in pioneering work by John Walker. However, obtaining a high-resolution structure of the intact ATP synthase is challenging because it is inherently dynamic. To overcome this conformational heterogeneity, we locked the yeast mitochondrial rotor in a single conformation by fusing a subunit of the stator with a subunit of the rotor, also called the central stalk. The engineered ATP synthase was expressed in yeast and reconstituted into nanodiscs. This facilitated structure determination by cryo–electron microscopy (cryo-EM) under near native conditions. RESULTS Single-particle cryo-EM enabled us to determine the structures of the membrane-embedded monomeric yeast ATP synthase in the presence and absence of the inhibitor oligomycin at 3.8- and 3.6-A resolution, respectively. The fusion between the rotor and stator caused a twisting of the rotor and a 9° rotation of the c 10 -ring, in the direction of ATP synthesis, relative to the putative resting state. This twisted conformation likely represents an intermediate state in the ATP synthesis reaction cycle. The structure also shows two proton half-channels formed largely by the a subunit that abut the c 10 -ring and suggests a mechanism that couples transmembrane proton movement to c 10 -ring rotation. The cryo-EM density map indicates that oligomycin is bound to at least four sites on the surface of the F o c 10 -ring that is exposed to the lipid bilayer; this is supported by binding free-energy molecular dynamics calculations. The sites of oligomycin-resistant mutations in the a subunit suggest that changes in the side-chain configuration of the c subunits at the a-c subunit interface are transmitted through the entire c 10 -ring. CONCLUSION Our results provide a high-resolution structure of the complete monomeric form of the mitochondrial ATP synthase. The structure provides an understanding of the mechanism of inhibition by oligomycin and suggests how extragenic mutations can cause resistance to this inhibitor. The approach presented in this study paves the way for structural characterization of other functional states of the ATP synthase, which is essential for understanding its functions in physiology and disease.