Crystal structure of subunits D and F in complex gives insight into energy transmission of the eukaryotic V-ATPase from Saccharomyces cerevisiae

Abstract

Eukaryotic V1VO-ATPases hydrolyze ATP in the V1 domain coupled to ion pumping in VO. A unique mode of regulation of V-ATPases is the reversible disassembly of V1 and VO, which reduces ATPase activity and causes silencing of ion conduction. The subunits D and F are proposed to be key in these enzymatic processes. Here, we describe the structures of two conformations of the subunit DF assembly of Saccharomyces cerevisiae (ScDF) V-ATPase at 3.1 Å resolution. Subunit D (ScD) consists of a long pair of α-helices connected by a short helix ((79)IGYQVQE(85)) as well as a β-hairpin region, which is flanked by two flexible loops. The long pair of helices is composed of the N-terminal α-helix and the C-terminal helix, showing structural alterations in the two ScDF structures. The entire subunit F (ScF) consists of an N-terminal domain of four β-strands (β1-β4) connected by four α-helices (α1-α4). α1 and β2 are connected via the loop (26)GQITPETQEK(35), which is unique in eukaryotic V-ATPases. Adjacent to the N-terminal domain is a flexible loop, followed by a C-terminal α-helix (α5). A perpendicular and extended conformation of helix α5 was observed in the two crystal structures and in solution x-ray scattering experiments, respectively. Fitted into the nucleotide-bound A3B3 structure of the related A-ATP synthase from Enterococcus hirae, the arrangements of the ScDF molecules reflect their central function in ATPase-coupled ion conduction. Furthermore, the flexibility of the terminal helices of both subunits as well as the loop (26)GQITPETQEK(35) provides information about the regulatory step of reversible V1VO disassembly.

Keywords: ATP Synthase; Bioenergetics; Proton Pump; Saccharomyces cerevisiae; Vacuolar ATPase.

FIGURE 1.

Arrangement of the existing individual atomic subunit structures in the EM map of the S. cerevisiae V-ATPase. Subunits C (1U7L; salmon), H (1HO8, brown), and F(1–94) (4IX9, blue) from S. cerevisiae were fitted into the EM map. The two conformations of EG subunits, the straight (4DL0; green and cyan) and more bent (4EFA; lemon and pale cyan) are fitted to the three peripheral stalks. Inset, region of the EM map showing the interaction of modeled subunit H (Ser-381) (yellow) through the sulfhydryl cross-linker 4-(N-maleimido)benzophenone (62) (stick; green) to the S. cerevisiae subunit F(1–94) (Glu-31). Left panel, schematic representation of the structures of the individual S. cerevisiae subunits C (1U7L; salmon), F(1–94) (4IX9, blue), H (1HO8, brown), and EG in two conformations, straight (4DL0; green and cyan) and bent (4EFA; lemon and pale cyan).

FIGURE 2.

Crystal structure of S. cerevisiae subunit DF complex. A, crystal structure of DF complex of the S. cerevisiae V-ATPase in an asymmetric unit; ScDF1 is shown in wheat and blue, and ScDF2 is shown in orange and light blue, respectively. B, schematic representation of the ScDF1 complex; ScD and ScF are shown in wheat and dark blue, respectively. Proline 179, which is conserved in all the eukaryotic ATP synthases, is labeled. C, schematic representation of ScF1 (dark blue), with the unique C terminus helix conformation and the α-helices and β-strands labeled. D, sequence alignment of subunit F from different ATPases. The secondary structure elements of subunit F from E. hirae and S. cerevisiae are shown. The conserved sequence in the eukaryotic ATP synthase is shown within the box. The ²⁶GQITPETQEK³⁵ loop of the F subunit is highlighted in red. The conserved Pro-89 is highlighted in red and is present in V-ATPases as well as A-ATP synthases. Proline 95, which is conserved in eukaryotic V ATPases, is highlighted by a green frame.

FIGURE 3.

Surface electrostatic and hydrophobicity of DF assembly and sequence comparison of subunit D. A, surface electrostatic potential of the ScDF complex with ScF shown as schematic and the DF interface labeled by an arrow is shown in the center. Surface electrostatic potential of ScD reveals that the interacting surface with ScF is mostly hydrophobic, whereas the exposed N and C termini of ScD are composed of basic and predominantly acidic residues (left). The interacting surface of ScF is predominantly hydrophobic (right). The electrostatic potential surfaces were calculated using APBS (56) and mapped at contouring levels from −3 kT (blue) to 3 kT (red). B, sequence alignment of subunit D from different V-ATPases and ATP synthases, respectively. The secondary structure elements of subunit D of the E. hirae A-ATP synthase and the S. cerevisiae V-ATPase are shown. The conserved sequence in the eukaryotic V-ATPases is highlighted in red.

FIGURE 4.

Intermolecular interactions of DF assembly. A, superimposition of the ScDF1 (wheat and blue) with ScDF2 molecule (orange and light blue). Residues Ala-155 and Pro-179 of subunit D and Pro-95 of subunit F are labeled. B, interactions in the ScDF1 molecule. The interaction of the very C terminus of ScF1 compact form with the N and C terminus of ScD1, and the interaction of the loop ²⁵IGQITPETQEK³⁵ of ScF1 with the loop between the short helix and β hairpin region of ScD1 are shown in the inset, respectively. C, ScDF2 molecule, where the ScF2 C-terminal helix is almost parallel to the N- and C-terminal helix of ScD2, is shown as schematic. Inset, interactions of the C-terminal helix of ScF2 is presented. The electron density omit map (contoured at 2σ) is shown in green mesh.

FIGURE 5.

Solution x-ray scattering studies of ScF. A, small angle x-ray scattering pattern (○) and its corresponding experimental fitting curves (- - -) for the concentrations 2.0 (black) and 4.2 (red) mg/ml. The scattering curves of ScF are displayed in logarithmic units for clarity. Inset, Guinier plots show linearity for the concentrations used, indicating no aggregation. B, pair-distance distribution function P(r) of ScF at 2.0 (black) and 4.2 (red) mg/ml. Inset, Kratky plot of ScF SAXS data at 4.2 mg/ml. C, fit of the scattering profile of the CORAL model with the best χ value of 1.24 (red) to the experimental scattering pattern of ScF at 2 mg/ml (black). D, CORAL model generated is superimposed with the solution shape of ScF. The compact monomer of the dimer is colored cyan, and the extended monomer is colored magenta. The C-terminal helices are colored lighter and the flexible regions are represented as dots and colored red.

FIGURE 6.

Fitting of the ScDF onto the DF subunits of the nucleotide-bound E. hirae A₁-ATP synthase structure (3VR6 (26)). A, extended C terminus of ScF (ScDF2 molecule) comes in contact with the EhA with the residue Lys-114 of ScF2 having electrostatic interaction with Asp-480 of EhA. Only one subunit A from E. hirae hexamer is shown for clarity. The nucleotide is shown in stick representation. B, close view of the various interactions of the extended ScF with EhA and B in the tight form of the A-B interface when ScDF2 is rotated by 80°. The α2 and α4 of ScF interacts with the C-terminal domain of EhA and α5 interacts with EhB. C, top view showing the C-terminal domain of subunit A and B from E. hirae A₁-ATP synthase and the position of ScDF2 after 80° rotation. ScF is in close proximity to the tight form of the nucleotide-binding site, and ScD lies between the empty and bound forms. D, interaction of ScD with subunits A and B of E. hirae. For clarity other subunits are removed from the hexamer.

FIGURE 7.

Superposition of subunit γ of the E. coli F-ATP synthase with ScF. Overall superposition of the E. coli subunit γ ((51) 3OAA; yellow) with ScF subunit (4RND; blue). Inset, globular domain of the γ subunit shares a similar fold with ScF. The α-helices and β-strands of ScF are labeled. The β-hairpin of Ecγ is shown in red. Two views of the superposition, rotated by 90°, are shown for clarity.