Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution

Abstract

The structure of the intact ATP synthase from the α-proteobacterium Paracoccus denitrificans, inhibited by its natural regulatory ζ-protein, has been solved by X-ray crystallography at 4.0 Å resolution. The ζ-protein is bound via its N-terminal α-helix in a catalytic interface in the F 1 domain. The bacterial F 1 domain is attached to the membrane domain by peripheral and central stalks. The δ-subunit component of the peripheral stalk binds to the N-terminal regions of two α-subunits. The stalk extends via two parallel long α-helices, one in each of the related b and b′ subunits, down a noncatalytic interface of the F 1 domain and interacts in an unspecified way with the a-subunit in the membrane domain. The a-subunit lies close to a ring of 12 c-subunits attached to the central stalk in the F 1 domain, and, together, the central stalk and c-ring form the enzyme's rotor. Rotation is driven by the transmembrane proton-motive force, by a mechanism where protons pass through the interface between the a-subunit and c-ring via two half-channels in the a-subunit. These half-channels are probably located in a bundle of four α-helices in the a-subunit that are tilted at ∼30°to the plane of the membrane. Conserved polar residues in the two α-helices closest to the c-ring probably line the proton inlet path to an essential carboxyl group in the c-subunit in the proton uptake site and a proton exit path from the proton release site. The structure has provided deep insights into the workings of this extraordinary molecular machine.Paracoccus denitrificans | ATP synthase | structure | regulation | proton translocation T he ATP synthases (F-ATPases) found in eubacteria, chloroplasts, and mitochondria are multiprotein molecular machines with a rotary action that provide most cellular ATP. Our understanding of how they work has come mainly from single-molecule studies of rotation made almost entirely on bacterial F-ATPases (1) and from structures of their constituent domains determined predominantly with enzymes from mitochondria (2-5). The most complete high-resolution structure contains about 85% of the bovine F-ATPase, built up from substructures determined by X-ray crystallography (2), within the constraints of an overall structure determined by cryo-EM (6). This model provides many details about the catalytic mechanism of the F 1 domain (2, 5); its mode of inhibition by the natural inhibitor protein of F 1 -ATPase, IF 1 (2, 4); and the design of the rotor, an ensemble of a membrane-bound ring of eight c-subunits, and the elongated central stalk, which penetrates into the catalytic F 1 domain. However, it lacks a crucial region that would help to explain how the enzyme uses the transmembrane proton-motive force produced by respiration or photosynthesis to generate the turning of the rotor in its membrane domain and other features that keep the proton-motive force coupled to the synthesis of ATP.Few structural studies have been carried out on the F-ATPases from eubacteria. Their subunit compositions are simpler than the subu...

Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

“…Cryo-EM and low-resolution X-ray crystallography have resolved these long and tilted α-helices in the F-type ATP synthase from the algae Polytomella sp. (19), the mammal Bos taurus (20), and the bacterium Paracoccus denitrificans (21). Identification of covarying pairs of residues in the sequences of homologous proteins, also known as evolutionary couplings, can identify pairs of residues that are likely to interact physically (22)(23)(24)(25).…”

“…A model was proposed for the a subunit from the mammalian ATP synthase using data from evolutionary covariance in conjunction with the cryo-EM density map (20). This atomic model of the B. taurus ATP synthase a subunit was shown to be accurate by an X-ray crystallography model of the a subunit from P. denitrificans that was published almost simultaneously with the B. taurus model, but which was not available when the modeling was done (21).…”

Biophysical comparison of ATP synthesis mechanisms shows a kinetic advantage for the rotary process

“…The rotary mechanism, on the other hand, may be viewed as a multistep process where the conformation changes incrementally with the transport of each proton. In F-ATPases, the number of protons transported varies from 8 to 15 per 360°rotation (yielding three ATPs) depending on cell and organelle type (21)(22)(23)48). The FE change that is possible in each step is limited by conformational and chemical constraints (31,54).…”

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

“…Because the sequences of the bovine and human c-subunits are identical, it is reasonable to assume that the human c-ring is identical. In the intact enzyme, the external surface of this ring is in contact with a single ATP6 (or a) subunit (15)(16)(17)(18). During ATP synthesis, the c 8 -ring turns with estimated speeds of up to 300 Hz.…”