The structure of the peripheral stalk of Thermus thermophilus H+-ATPase/synthase

Abstract

Proton-translocating ATPases are ubiquitous protein complexes that couple ATP catalysis with proton translocation via a rotary catalytic mechanism. The peripheral stalks are essential components that counteract torque generated from proton translocation during ATP synthesis or from ATP hydrolysis during proton pumping. Despite their essential role, the peripheral stalks are the least conserved component of the complexes, differing substantially between subtypes in composition and stoichiometry. We have determined the crystal structure of the peripheral stalk of the A-type ATPase/synthase from Thermus thermophilus consisting of subunits E and G. The structure contains a heterodimeric right-handed coiled coil, a protein fold never observed before. We have fitted this structure into the 23 A resolution EM density of the intact A-ATPase complex, revealing the precise location of the peripheral stalk and new implications for the function and assembly of proton-translocating ATPases.

Figure 1

Architecture and nomenclature of the intact T. thermophilus A-ATPase/ synthase. (a) Side view of the A-ATPase. The stator subunits (A, B, E, G and I) are labeled in white and the rotor subunits (D, F, C and L) are labeled in black. (b) View from above. The black arrow indicates the rotational direction of the central stalk during ATP synthesis and the white arrows indicate the torque in the A₃B₃ nucleotide-binding domain that subunit E counteracts.

Figure 2

Structure of the EG peripheral stalk complex. (a) Ribbon representation with subunit E (blue to cyan) from N to C terminus and subunit G (green). The C termini of both subunits are shaded in gray and form a globular head group, whereas the N-terminal helices form an RHCC. These domains are tethered via flexible loops, one containing three proline residues (3× Pro loop) in subunit E and the other consisting of a kink at Arg106 in subunit G. Bottom, N-terminal residues of subunits E (Ser2) and G (Gly21). (b) Surface representation of the complex rotated clockwise by 90° (same colors as in a).

Figure 3

Hydrophobic repeats forming the RHCC interface between subunits E and G. (a,b) The sequence of subunit E (a) and subunit G (b), formatted to highlight the hendecad and quindecad hydrophobic repeats that mediate the interaction between the two subunits. Residues at the intersubunit interface are highlighted in yellow; residues forming intersubunit hydrogen bonds or salt bridges are in red. Purple highlighting of Arg106 marks the position of the kink in chain G. (c) Surface representation of the EG dimer with subunit E in blue, subunit G in green and residues at the intersubunit interface in yellow. Below, regions of the RHCC with a hendecad and quindecad repeat in subunit G.

Figure 4

Superposition of the P. horikoshii subunit E C-terminal domain and T. thermophilus subunit E. The C-terminal domain of subunit E from P. horikoshii (PDB accession code 2dma) is presented in yellow to red from the N to C terminus; T. thermophilus subunit E is presented in dark blue to light blue from the N to C terminus; and T. thermophilus subunit G is in green. Top, location of the three-proline loop of T. thermophilus subunit E, which forms a 90° bend in between helices 1 and 2, whereas the angle in between P. horikoshii helices 1 and 2 is closer to 60°. Left, Arg106, which forms a 50° kink in subunit G.

Figure 5

Docking of the E–G peripheral stalk complex into the 23-Å EM density of the intact T. thermophilus A-ATPase/synthase. (a–c) The EM reconstruction with only the EG peripheral stalk docked in is shown from the side (a), top (b) and a vertical cross-section just below the A₃B₃ head group (c). (d–f) EM reconstruction plus a model (in gray) of the X-ray structures of the T. thermophilus A₁ complex (A₃B₃DF (PDB accession code 3a5c), T. thermophilus subunit C⁴⁶ (PDB accession code 1r5z) and a model of 12 E. coli subunit c protomers based on NMR data⁴⁷ (PDB accession code 1c17). Residue Lys25 from subunit B is indicated by red spheres. The coordinates of the composite model of these structures are available in the Supplementary Data. (d) The eukaryotic V-ATPase subunit C (Vma5p; PDB accession code 1u7l) is docked (in purple) to demonstrate its complementarity in size and shape to the soluble part of subunit I. (e,f) Close-up view of the distal (e) and proximal (f) E–G stator head group fitted into the EM density. Gray arrowheads indicate deviations of the EG structure from the EM density.