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 F1 domain. The bacterial F1 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 F1 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 F1 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.

Keywords: ATP synthase; Paracoccus denitrificans; proton translocation; regulation; structure.

Fig. 1.

Structure of the complex of the F-ATPase from P. denitrificans with the bound ζ-inhibitor protein. (A and B) Side views of the enzyme–inhibitor complex in surface representation. B is rotated right by 90° relative to A. (Upper) Membrane extrinsic F₁ catalytic domain (red, yellow, blue, and green corresponding to three α-subunits, three β-subunits, and single γ- and ε-subunits, respectively). In the peripheral stalk, the δ-subunit (top) is sky blue and the long and approximately parallel α-helical segments in orange and pink (chains V and W) extending down the surface of the interface between the α- and β-subunits are parts of the b- and b′-subunits (undistinguished). Unassigned α-helical segments (chains 1 and 2) in the vicinity of the junction between the δ-subunit and b- and b′-subunits are purple and light gray, respectively. Helix-1 of the ζ-inhibitor is brown. (Lower) In the membrane domain, the ring of 12 c-subunits is gray and a bundle of four resolved α-helices assigned to the a-subunit is lemon green. An unassigned α-helical segment in magenta (chain 3) lies approximately parallel to α-helices in subunit a, and two unassigned side-by-side transmembrane α-helices (chain Y) are colored light blue.

Fig. 2.

Mode of binding of the ζ-inhibitor to the F-ATPase from P. denitrificans. The inhibitor is bound in the α_DPβ_DP-catalytic interface of the enzyme. (A) Cross-sectional side view of the F₁ domain showing the interaction of the ζ-inhibitor protein (brown) with the C-terminal domain of the β_DP-subunit (yellow) and the coiled-coil of α-helices in the γ-subunit (blue). (B) View from outside the complex toward the α_DPβ_DP-catalytic interface with the N-terminal α-helix of the ζ-inhibitor in a cleft between the α_DP- and β_DP-subunits. (C) Potential interactions between side chains of the ζ-inhibitor protein with residues in the α_DP-, β_DP-, and γ-subunits (Table S2). (D) Composite structure of the ζ-inhibitor by combination of residues 1–32 and 82–103 from the current study (brown) with residues 15–104 of the solution structure (cyan). (E) Superposition of the N-terminal region of the ζ-inhibitor (brown) with the corresponding inhibitory regions of IF₁ from bovine and yeast mitochondria (cyan and pink, respectively). The α-helical regions are residues 21–49, 16–36, and 3–24, respectively.

Fig. S1.

Alignment of the N-terminal sequence of the ζ-inhibitor from P. denitrificans with the N-terminal sequences of F-ATPase inhibitor proteins bIF₁ and yIF₁ from bovine and yeast mitochondria, respectively. Green, red, blue, and ochre indicate residues that are polar but uncharged, acidic, basic, and hydrophobic, respectively.

Fig. 3.

Interactions of the δ-subunit with N-terminal regions of α-subunits in the F-ATPase from P. denitrificans. (A) View from above the F-ATPase toward the “crown” of the F₁ domain depicting the N-terminal regions of the α_E-, α_TP-, and α_DP-subunits (red) with the δ-subunit (blue), β-subunits (yellow) and chains 1 and 2 (Ch1 and Ch2; purple and gray, respectively). (B) Side view of the interactions of the α_E-subunit (residues 7–22) and the α_TP-subunit (residues 2–22) with helices δH1 and δH5 and helices δH2, δH3, and δH4, respectively. (C) Side view of the region around the N-terminal part of the α_DP-subunit with structural elements from peripheral stalk subunits.

Fig. 4.

Topography of the membrane domain of the F-ATPase from P. dentrificans and a potential pathway of transmembrane proton translocation. (A) View of the c₁₂-rotor ring, and an associated bundle of α-helices (green), assigned to the a-subunit and named aH3–aH4 (residues 1,001–1,035), and aH5 and aH6 containing residues 166–198 and 217–246, respectively (Figs. S2 and S3). Unassigned α-helix Ch3 and α-helical hairpin ChY are shown in magenta and blue, respectively. (B) View of the association of the tilted bundle of four α-helices in subunit a with the c-ring showing residue Glu60 (red) in the c-subunit in the proton transfer site and residue Arg182 (blue) in aH5. (C) View from the c-ring of the tilted bundle of four α-helices in subunit a showing conserved polar residues (yellow) that could provide the access path (In) for protons from the bacterial periplasm to reach the proton transfer site and the exit path (Out) for protons to be released into the bacterial cytoplasm. Residue Arg182 is colored blue. (D) View in solid representation of the tilted bundle of four α-helices in the a-subunit in juxtaposition with the c-ring showing the potential inlet pathway for protons (yellow) leading through the bundle to the proton transfer site containing a negatively charged Glu60 (red).

Fig. S2.

Predicted transmembrane α-helices in the b-, b′-, and a-subunits of the F-ATPase from P. denitrificans. For comparison, the predictions for b- and a-subunits from E. coli and for the b′-subunit from Spinacea oleracea are shown also. The predictions were calculated by TMHMM.

Fig. S3.

Alignment of sequences of a-subunits. The sequences cover a wide range of species. The arrow denotes the position of the Arg residue (Arg210 in E. coli, Arg182 in P. denitrificans) that is an essential part of the transmembrane proton translocation pathway.