Cryo-EM structures of the autoinhibited E. coli ATP synthase in three rotational states.

Meghna Sobti,Andrew Sw Wong,Robert Ishmukhametov,Sara Sandin,Daniela Stock,Alastair G Stewart,Callum Smits

doi:10.7554/elife.21598

Abstract

A molecular model that provides a framework for interpreting the wealth of functional information obtained on the E. coli F-ATP synthase has been generated using cryo-electron microscopy. Three different states that relate to rotation of the enzyme were observed, with the central stalk's ε subunit in an extended autoinhibitory conformation in all three states. The Fo motor comprises of seven transmembrane helices and a decameric c-ring and invaginations on either side of the membrane indicate the entry and exit channels for protons. The proton translocating subunit contains near parallel helices inclined by ~30° to the membrane, a feature now synonymous with rotary ATPases. For the first time in this rotary ATPase subtype, the peripheral stalk is resolved over its entire length of the complex, revealing the F1 attachment points and a coiled-coil that bifurcates toward the membrane with its helices separating to embrace subunit a from two sides.

Highlights

In most cells, the bulk of ATP, the principal source of cellular energy, is synthesized by ATP synthase
The Fo motor spans the membrane converting the potential energy of the proton motive force into rotation of the central stalk that in turn drives conformational changes in the F1 catalytic sites
Cysteine-free E. coli F-ATPase, as described in Ishmukhametov et al (2005) where all 10 cysteines were replaced with alanines and a His-tag introduced on the b subunit, was solubilized in digitonin detergent and purified as described in the Materials and methods

Summary

Introduction

The bulk of ATP, the principal source of cellular energy, is synthesized by ATP synthase This molecular generator couples ion flow across membranes with the addition of inorganic phosphate (Pi) to ADP thereby generating ATP (Iino and Noji, 2013; Stewart et al, 2014). Recent sub nanometer electron cryo-microscopy (cryo-EM) reconstructions of F-type (Allegretti et al, 2015; Zhou et al, 2015; Kuhlbrandt and Davies, 2016; Hahn et al, 2016) and the analogous V- and A-type ATPases (Zhao et al, 2015; Schep et al, 2016) as well as a low-resolution crystal structure of Paracoccus denitrificans F-ATPase (MoralesRios et al, 2015) are consistent with a two half-channel mechanism for the generation of rotation within the membrane (Vik and Antonio, 1994; Junge et al, 1997).

Methods

Results

Conclusion