CryoEM Reveals the Complexity and Diversity of ATP Synthases

Abstract

During respiration, adenosine triphosphate (ATP) synthases harness the electrochemical proton motive force (PMF) generated by the electron transport chain (ETC) to synthesize ATP. These macromolecular machines operate by a remarkable rotary catalytic mechanism that couples transmembrane proton translocation to rotation of a rotor subcomplex, and rotation to ATP synthesis. Initially, x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cross-linking were the only ways to gain insights into the three-dimensional (3D) structures of ATP synthases and, in particular, provided ground-breaking insights into the soluble parts of the complex that explained the catalytic mechanism by which rotation is coupled to ATP synthesis. In contrast, early electron microscopy was limited to studying the overall shape of the assembly. However, advances in electron cryomicroscopy (cryoEM) have allowed determination of high-resolution structures, including the membrane regions of ATP synthases. These studies revealed the high-resolution structures of the remaining ATP synthase subunits and showed how these subunits work together in the intact macromolecular machine. CryoEM continues to uncover the diversity of ATP synthase structures across species and has begun to show how ATP synthases can be targeted by therapies to treat human diseases.

Keywords: ATP synthase; bioenergetics; cryoEM; membrane; protein; structure.

Figure 1

Subunit composition of commonly studied adenosine triphosphate (ATP) synthases. (A) Cartoon of a bacterial ATP synthase. Based on Guo et al. (2019). (B) Cartoon of a mammalian mitochondrial ATP synthase. IMS, intermembrane space.

Figure 2

Rotary catalytic mechanism of ATP synthase. (A) Atomic model of Bacillus PS3 ATP synthase showing the three main rotational states (PDB: 6N2Y, 6N30, and 6N2Z) (Guo et al., 2019). The light blue parts of the complex rotate relative to the dark blue parts. The direction of rotation during ATP hydrolysis is indicated. (B) Diagram of the catalytic cycle of the F₁ region during ATP hydrolysis. (C) Structures of the Bacillus PS3 F₁ complex catalytic substeps (PDB: 7L1R and 7L1Q) (Sobti et al., 2021). White bars represent the angle of the γ subunit. ATP, ADP, and non-catalytic nucleotides are colored orange, green, and dark gray, respectively. Based on Sobti et al. (2021).

Figure 3

Proton translocation in ATP synthase. (A) Proton path (blue arrow) through the interface between the a and c subunits of the F_O region of bovine ATP synthase (PDB: 6ZPO) (Spikes et al., 2020). The conserved arginine in subunit a and acidic residues in the c ring are shown as space filling models. (B) Electrostatic surface of bovine subunit a, with positively and negatively charged surfaces colored blue and red, respectively.

Figure 4

Diversity of ATP synthases. Atomic models of ATP synthases from diverse species. Homologs of subunits α, β, γ, δ, ε, and c are colored red, yellow, blue, pink, purple, and gray, respectively. From the left to right, top to bottom: Mycobacterium smegmatis (PDB: 7JG5) (Guo et al., 2021), Saccharomyces cerevisiae dimer (PDB: 6B8H) (Guo et al., 2017), Polymotella sp. dimer (PDB: 6RD4) (Murphy et al., 2019), Euglena gracilis dimer (PDB: 6TDU) (Mühleip et al., 2019), Spinacia oleracea (PDB: 6FKF) (Hahn et al., 2018), Sus scofa domesticus tetramer (PDB: 6J5K, 6ZNA) (Gu et al., ; Spikes et al., 2020), Tetrahymena thermophila tetramer (PDB: 6YNZ) (Flygaard et al., 2020), and Toxoplasma gondii hexamer (PDB: 6TML) (Mühleip et al., 2021).

Figure 5

Structures of drug-bound ATP synthases. (A) Atomic model of M. smegmatis ATP synthase bound to the tuberculosis (TB) drug bedaquiline (BDQ) (PDB: 7JGC) (Guo et al., 2021). Bedaquiline binds at five c-only sites (yellow), a leading site (pink), and a lagging site (blue) in the F_O region of the enzyme. Red arrows indicate the movement of residues upon bedaquiline binding. (B) Atomic model of S. cerevisiae ATP synthase F₁ region bound to Ammocidin (PDB: 7MD2) (Reisman et al., 2021). Ammocidin (green) binds at the rotor–stator interface (black arrow).