The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF₁

Abstract

The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α(DP)- and β(DP)-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β(E)-subunits. The nucleotide binding site in β(E)-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.

Figure 1.

Alignment of the sequences of residues 1–60 of bovine IF₁, and the equivalent region of yeast IF₁, with the same regions from other species. The purple, green and yellow stripes denote identical, highly conserved and poorly conserved residues, respectively. The alignment was performed with ClustalW. The bars above the sequences denote α-helical regions in the bovine protein. The yIF₁ used in crystallization experiments contained the mutation E21A.

Figure 2.

Gel filtration chromatography of yeast and bovine F₁-ATPase-IF₁ complexes. The yeast and bovine enzymes were inhibited with the inhibitor protein from S. cerevisiae (yF₁) and with bovine IF₁ (bIF₁), respectively. (a) Column profiles of yF₁–yIF₁, active yF₁ and bF₁–bIF₁ complexes, respectively. V₀ is the void volume of the column. (b) SDS–PAGE analysis of peaks 1, 2 and 3 from (a).

Figure 3.

The structure of the F₁-I1–53 complex from S. cerevisiae. The α-, β-, γ-, δ- and ε-subunits are depicted in ribbon form in red, yellow, dark blue, green and magenta, respectively, and residues 1–36 of I1–53 are light blue. (a) Overall view of the complex viewed from the side with IF₁ shown in solid representation. (b) View (upwards from the foot of the central stalk) along the axis of the γ-subunit showing the position of the α-helix of yeast IF₁ in ribbon representation relative to the C-terminal domains of α- and β-subunits.

Figure 4.

The binding site for yeast I1–53 in the structure of yeast F₁-ATPase. (a) The N-terminal loop region of the inhibitor protein (light blue) in juxtaposition with the C-terminal helix of the γ-subunit (dark blue). Dotted lines represent possible interactions with distances in angstrom. (b) View from the side of the central stalk showing the orientations of the yeast and bovine inhibitor proteins (light blue and brown, respectively) relative to the central stalk. (c) View from outside the F₁-domain towards the γ-subunit of the enzyme (dark blue) of the deep cleft between the C-terminal domains of the α_DP- and β_DP-subunits (red and yellow, respectively) where the α-helical region of I1–53 (light blue) is bound. The position of the equivalent region of the bovine inhibitor protein in the structure of bovine F₁-I1–60 is shown in brown. (d) View of the bovine inhibitor (brown) superposed onto the yeast inhibitor protein (light blue) via residues 22–25 and 17–20 in the bovine and yeast inhibitors, respectively.

Figure 5.

Influence of point mutation of selected residues in yI1–53 on its inhibition of yeast F₁-ATPase. The quantitative data from which the figure is derived are given in table 2. K_iwt and K_imut are the dissociation constants for the wild-type and mutant proteins, respectively.

Figure 6.

Change in the coordination of a magnesium ion in the active site of yeast F₁-ATPase. (a) Hexacoordination of the magnesium ion (green) in the catalytic site of the β_DP-subunit. The ligands are provided by water molecule a–d (red), by the oxygen atom βO2 of ADP (or ATP), and by the β-hydroxy-group of β_DP-T164. The water molecules a–d are themselves hydrogen-bonded to other water molecules e–g (grey), by the oxygen atom βO1 of the nucleotide, and by the side chain functionalities of residues β_DP-E189, β_DP-R190 and β_DP-D256. (b) Schematic of the disposition of the magnesium ion and bound ADP in the catalytic site of the β_DP-subunit. The P-loop sequence (upper left) helps to bind the magnesium ion and the nucleotide. The adenosine moiety of the nucleotide is bound in the hydrophobic pocket between the side chains of residues β_DP-Y345 and β_DP-F424. Distances are given in angstrom, and those to the phosphate group are to the closest oxygen atom. (c) Rotation of γ-subunit driven by hydrolysis of ATP has opened the nucleotide binding domain of the subunit, converting the β_DP-site to the β_E-site. The catalytic ‘arginine finger’ residue, αR375 becomes disordered, and the coordination sphere of the magnesium ion is disrupted, releasing the metal ion. In the structure of yF₁-I1–53, the inhibitor protein has arrested the conversion of the site just before the formation of a fully ‘empty’ or ‘open’ site. The adenosine binding pocket is still intact, and ADP remains bound to the subunit. In the final step of the conversion of the site to a fully formed β_E-site (as observed in ‘ground state’ structures of F₁-ATPase), the side chain of β_E-F424 rotates through 90°, releasing the ADP molecule.

Figure 7.

Comparison of the nucleotide binding pockets in the β_E-subunits in the structures of yF₁-I1–53 and bovine F₁-PH. The yeast and bovine protein backbones are coloured yellow and pink, respectively. The side chains of residues β_E-F424 and β_E-Y345 are red in the yeast enzyme and pink in the bovine enzyme. They provide the pocket for binding the adenosine moieties of the ADP molecules (blue and pink, respectively in the yeast and bovine enzymes). In grey is shown α-helix b (residues 102–110) in the δ-subunit of an adjacent yeast F₁-complex in the crystal lattice of yeast F₁-I1–53. It approaches to within 4 Å of α-helix C3 carrying β_E-F424 in the yeast structure. Thus, it makes a crystal contact that may influence the position of α-helix C3 in the β_E-subunit of the yeast enzyme.