You are currently viewing the abstract.
View Full TextLog in to view the full text
AAAS login provides access to Science for AAAS members, and access to other journals in the Science family to users who have purchased individual subscriptions.
More options
Download and print this article for your personal scholarly, research, and educational use.
Buy a single issue of Science for just $15 USD.
Protons find a path
Adenosine triphosphate (ATP) synthases are dynamos that interconvert rotational and chemical energy. Capturing the complete structure of these multisubunit membrane-bound complexes has been hindered by their inherent ability to adopt multiple conformations. Srivastava et al. used protein engineering to freeze mitochondrial ATP synthase from yeast in a single conformation and obtained a structure with the inhibitor oligomycin, which binds to the rotating c-ring within the membrane. Hahn et al. show that chloroplast ATP synthase contains a built-in inhibitor triggered by oxidizing conditions in the dark chloroplast. The mechanisms by which these machines are powered are remarkably similar: Protons are shuttled through a channel to the membrane-embedded c-ring, where they drive nearly a full rotation of the rotor before exiting through another channel on the opposite side of the membrane (see the Perspective by Kane).
Science, this issue p. eaas9699, p. eaat4318; see also p. 600
Structured Abstract
INTRODUCTION
Green plant chloroplasts convert light into chemical energy, and adenosine triphosphate (ATP) generated by photosynthesis is the prime source of biologically useful energy on the planet. Plants produce ATP by the chloroplast F1Fo ATP synthase (cF1Fo), a macromolecular machine par excellence, driven by the electrochemical proton gradient across the photosynthetic membrane. It consists of 26 protein subunits, 17 of them wholly or partly membrane-embedded. ATP synthesis in the hydrophilic α3β3 head (cF1) is powered by the cFo rotary motor in the membrane. cFo contains a rotor ring of 14 c subunits, each with a conserved protonatable glutamate. Subunit a conducts the protons to and from the c-ring protonation sites. The central stalk of subunits γ and ε transmits the torque from the Fo motor to the catalytic cF1 head, resulting in the synthesis of three ATP per revolution. The peripheral stalk subunits b, b′, and δ act as a stator to prevent unproductive rotation of cF1 with cFo.
All rotary ATP synthases are, in principle, fully reversible. To prevent wasteful ATP hydrolysis, cF1Fo has a redox switch that inhibits adenosine triphosphatase (ATPase) activity in the dark.
RATIONALE
Understanding the molecular mechanisms of this elaborate nanomachine requires detailed structures of the whole complex, ideally at atomic resolution. Because of the dynamic nature of this membrane protein complex, crystallization has been difficult and no high-resolution structure of an entire, functional ATP synthase is available. We reconstituted cF1Fo from spinach chloroplasts into lipid nanodiscs and determined its structure by cryo–electron microscopy (cryo-EM). Cryo-EM is the ideal technique for this study because it can deliver high-resolution structures of large, dynamic macromolecular assemblies that adopt a mixture of conformational states.
RESULTS
We present the cryo-EM structure of the intact cF1Fo ATP synthase in lipid nanodiscs at a resolution of 2.9 Å (cF1) to 3.4 Å (cFo). In the cF1 ATPase head, we observe nucleotides with their coordinating Mg ions and water molecules, allowing assignment to the three well-characterized functional states involved in rotary ATP synthesis. Subunit δ on top of the ATPase headbinds to all three α subunits, ensuring that only one peripheral stalk can attach. The loosely entwined, long α helices of the peripheral stalk subunits b and b′ clamp the integral membrane subunit a in its position next to the c-ring rotor, thus connecting cF1 to cFo. Subunit γ has an L-shaped double hairpin with a redox sensor that can form a disulfide bond and a chock that blocks rotation to avoid wasteful ATP hydrolysis at night.
Protons are translocated through access routes in subunit a in all rotary ATPases. We observe a hydrophilic channel on the lumenal surface that connects to the glutamate residues on the c-ring rotor that carry protons for an almost full rotation before releasing them into the stroma through another hydrophilic channel. A strictly conserved arginine separates the access and exit channels, preventing leakage of protons through the membrane.
CONCLUSION
We observe three cF1Fo conformations, each with the central rotor stalled in a different position. Ring rotation is unexpectedly divided into three unequal steps. The peripheral stalk may thus act like an elastic spring, evening out the different energy contributions of each step. The features of ATP synthase nanomachines are remarkably similar in chloroplasts and mitochondria, considering their evolutionary distance of a billion years or more.
Subunits α and β contain the nucleotide-binding sites with resolved nucleotides, Mg2+, and water molecules. Subunit δ joins cF1 to the membrane-embedded motor via the peripheral stalk (b, b′) that positions subunit a against the rotor ring. The electrochemical proton gradient drives ring rotation (arrow). The central stalk (γε) transmits torque to cF1. The redox regulator blocks rotation in the dark.
Abstract
The chloroplast adenosine triphosphate (ATP) synthase uses the electrochemical proton gradient generated by photosynthesis to produce ATP, the energy currency of all cells. Protons conducted through the membrane-embedded Fo motor drive ATP synthesis in the F1 head by rotary catalysis. We determined the high-resolution structure of the complete cF1Fo complex by cryo–electron microscopy, resolving side chains of all 26 protein subunits, the five nucleotides in the F1 head, and the proton pathway to and from the rotor ring. The flexible peripheral stalk redistributes differences in torsional energy across three unequal steps in the rotation cycle. Plant ATP synthase is autoinhibited by a β-hairpin redox switch in subunit γ that blocks rotation in the dark.
This is an article distributed under the terms of the Science Journals Default License.
