Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase

Abstract

The crystal structure of yeast mitochondrial F(1) ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the alpha(3)beta(3) sub-assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F(1) ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F(1) ATPase has an ATP analog bound and therefore this structure does not represent the ADP-inhibited form. In addition, one of the complexes binds phosphate in the nucleotide-free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F(1) ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis.

Figure 1

Comparison of the α/β catalytic interfaces in bovine and yeast F₁ ATPase. Stereo image (wall-eyed) of the α-carbon traces of the α- (red) and β- (blue) subunits of the yeast DP-II overlaid on the corresponding subunits of BeF₃⁻-inhibited bovine F₁ (gray) (top) and DP-I overlaid on DP-II (salmon) (bottom). The molecules were superimposed as the α/β pair using TOP3D (Collaborative_Computational_Project, 1994). The nucleotide bound to the β_DP-subunit is shown in space-filling representation. The abbreviations are: DP-I, the α_DP/ β_DP interface from yF₁I; DP-II, the α_DP/ β_DP interface from yF₁II.

Figure 2

Comparison of the β_DP active sites of yeast and bovine F₁ ATPase stereo image (wall-eyed) of the active site of the yeast DP-I (gold) superimposed with the β_DP site of the ADP:BeF₃⁻-inhibited bovine enzyme (white) (Kagawa et al, 2004). The bovine residue numbering is shown in brackets when it differs from the yeast numbering. BeF₃ is colored yellow and phosphate is colored magenta. The Mg²⁺ ion is shown as a sphere, colored gold in the bovine structure and red in yF₁.

Figure 3

Phosphate-binding site in the β_E-subunit of the yF₁II complex. (A) Electron density of the final 2F_o-F_c map for the phosphate-binding site (contoured at 1.3σ). The electron density is shown only for a radius about the phosphate to simplify the image. (B) Side chains that contribute to phosphate binding. Possible ionic interactions are shown as dotted lines, with distances in Å. (C) Superposition of the phosphate-binding region of yF₁I (green) on that of yF₁II (blue). (D) Superposition of the phosphate-binding region of the empty subunit of bovine F₁ (pink) on yF₁II (blue). The bovine residue numbering is used in this image.

Figure 4

Structure of the central stalk of yeast F₁ ATPase. The central stalk subunits of yeast yF₁I, γ-(green), δ-(slate blue), and ɛ-(purple) subunits are superimposed on the bovine (gold) or yeast yF₁II (red). (A) The central stalks of bovine F₁ and the yeast yF₁I complex were superimposed using TOP3D to illustrate the overall similarity of the fold. (B) The central stalks of bovine F₁ and the yeast yF₁I complex were superimposed using the N-terminal β-barrel domains of the α- and β-subunits to illustrate the relative position within the core of F₁. (C) The γ-subunit from the yF₁I complex is shown superimposed on that of the yF₁II complex using the N-terminal β-barrel domains of the α- and β-subunits. (D) Comparison of the δ-subunits of the yeast and bovine enzymes. (E) Comparison of the ɛ-subunits of the yeast and bovine enzymes. (F) Stereo image (wall-eyed) illustrating the rotation of the γ-subunit from yF₁I (green) to yF₁II (magenta) showing residues 4–35 and 241–276. Stereo images of A–C are in the supplement.

Figure 5

Relative movement of the phosphate molecule during the catalytic cycle. The predicted path of the phosphate molecule during catalysis is marked by the position of phosphate (or sulfate) in the β_E-subunits of the yF₁II complex (blue), the bovine AlF₄⁻:ADP-inhibited structure (Menz et al, 2001) (yellow), and the γ-phosphate of AMPPNP bound to the β_DP-subunit of the yF₁I complex (salmon). The structures were superimposed using the P-loop and neighboring catalytic residues (β151–177, β330–336). The α-carbon trace of the P-loop of all three enzymes is shown along with the bound nucleotide and phosphate (or sulfate) of yF₁II (yellow). The inset shows just the movement of the phosphate relative to the nucleotide. The phosphate bound to β_E (blue) moves to the position in the AlF₄⁻:ADP-inhibited state (yellow) and ends as the γ-phosphate of ATP in the DP site (as colored). Also shown is the movement of αArg375 in the same path. The distances between the atoms are shown in Å.