Journal Article

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

Rohou, Alexis, Schep, Daniel G, Walker, John E, Zhou, Anna, Bason, John V, Rubinstein, John L, Montgomery, Martin G, Grigorieff, Nikolaus

2015

Overview

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. A molecule called adenosine triphosphate (ATP) is the energy currency in cells. Most of the ATP used by cells is made by the membrane-embedded enzyme ATP synthase. This enzyme is found in membranes inside specialized compartments known as mitochondria. ATP synthase is made up of many protein subunits that work together as a molecular machine. Hydrogen ions flow across the membrane through the ATP synthase, turning a rotor structure within the enzyme, which leads to the production of ATP. It is not known how the transport of hydrogen ions causes rotation of the rotor. Some researchers have proposed that the enzyme works as a ratchet that is driven by the random Brownian motion of the rotor. That is, the rotational position of the rotor fluctuates randomly, but a ratchet mechanism ensures that there is a net rotation in one direction. However, there is currently little experimental evidence to back up this theory, which is known as the Brownian ratchet model. Zhou, Rohou et al. used a technique called electron cryomicroscopy (or cryo-EM) to study ATP synthase from cows. The cryo-EM data made it possible to use computer software to construct a three-dimensional model of the enzyme that is more detailed than previous attempts. Zhou, Rohou et al. show that the structure of ATP synthase is flexible, with the different protein subunits bending, flexing, and rotating relative to each other. This variability in the position of the rotor is consistent with the Brownian ratchet model. Together, these findings reveal important new details about the structure of ATP synthase and provide some of the first experimental evidence for the Brownian ratchet model. The new three-dimensional structure of ATP synthase will open the door to testing hypotheses of how the ATP synthase works.

• Publisher: eLife Science Publications, Ltd,eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Adenosine triphosphate / Animals / ATP synthase / Biochemistry / Biophysics and Structural Biology / bovine / Cattle / Classification / coevolution / Computational Biology / cryo-EM / Cryoelectron Microscopy / Data processing / Enzymes / evolutionary covariance / Imaging, Three-Dimensional / Lipids / Methods / Mitochondria / Mitochondrial Proton-Translocating ATPases - chemistry / Mitochondrial Proton-Translocating ATPases - genetics / Models, Molecular / Physiological aspects / Protein Conformation / Protein Folding / Saccharomyces cerevisiae / Structure