Ground State Structure of F1-ATPase from Bovine Heart Mitochondria at 1.9 Aå Resolution

Abstract

The structure of bovine F 1 -ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9 Å resolution. This structure has been compared with the previously described structure of bovine F 1 -ATPase determined at 1.95 Å resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the ␤ DPsubunit. In the present structure, the nucleotide binding sites in the ␤ DP -and ␤ TP -subunits are both occupied by AMP-PNP, whereas in the earlier structure, the ␤ TP site was occupied by AMP-PNP and the ␤ DP site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, ␣Arg-373 differs in the ␤ DP -and ␤ TP -subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.Our current understanding of the molecular mechanism of F 1 -ATPase is based on the structural analysis by x-ray crystallography of the enzyme from bovine heart mitochondria. The first high resolution structure (1), now known as the "reference" structure, determined at 2.8 Å resolution with crystals grown in the presence of both ADP and the nonhydrolyzable ATP analog AMP-PNP, 3 showed that the three noncatalytic ␣-subunits and the three catalytic ␤-subunits are arranged in alternation around an asymmetric ␣-helical structure in the single ␥-subunit. The ␣-and ␤-subunits have similar folds consisting of an N-terminal domain with six ␤-strands, a central nucleotide binding domain made of both ␣-helices and ␤-strands and an ␣-helical C-terminal domain containing six ␣-helices in ␤-subunits and seven in ␣-subunits. Because of the asymmetry of the ␥-subunit, the catalytic ␤-subunits adopt different conformations with different nucleotide occupancies. Two of them have similar conformations, but one, designated as ␤ DP , contains bound ADP, and the second, ␤ TP , has bound AMP-PNP. The third has adopted a radically different conformation in which the nucleotide binding domain has been disrupted by an outward hinging movement of part of the domain and the attached C-terminal domain in response to the curvature of the central ␣-helical structure of the ␥-subunit. This ␤-subunit has no bound nucleotide, and so it is known as the "empty" or open state, designated as ␤ E . To explain the interconversion of catalytic sites through "tight," "loose," and "open" states required by a binding change mechanism of catalysis of ATP hydrolysis by F 1 -ATPase (2), it was proposed that the interconversion of sites is effected by a mechanical rotation of the ␥-subunit, each 360°r otation taking each ␤-subunit through the three states and thereby hydrolyzing three ATP molecules. It was shown subsequently that during ATP hydrolysis, either in an ␣ 3 ␤ 3 ␥ complex or in the...

How the regulatory protein, IF 1 , inhibits F 1 -ATPase from bovine mitochondria

“…The diffraction properties of crystals of the F 1 -I1-60His complex were improved by shrinking the unit cell, especially in the a dimension, by changing the relative humidity from 98.5% to 98.0-97.5% by controlled dehydration. Similar benefits derived from controlled dehydration of crystals of bovine F 1 -ATPase grown in the presence and absence of azide have been described (6,14). Quaternary structural changes relative to the reference (14,15) and (F 1 -IF 1 ) 2 structures can be attributed to increased lattice contacts (see SI Text).…”

“…2 C and D). At this point, the chain turns abruptly right (as viewed from the side of the F 1 particle), at almost 90°, allowing the linker (residues [19][20] between the short and long helices, and the short helix itself (residues [14][15][16][17][18], to make more contacts with the N-terminal helix of the ␥-subunit. The unstructured region (residues 8-13) preceding the short helix snakes around the N-terminal ␣-helix of the ␥-subunit in an anticlockwise direction (as viewed from the mitochondrial membrane), emerges into the central cavity, and crosses it, making contacts with the nucleotide binding domain of the ␣ E -subunit (Fig.…”

“…In the F 1 -I1-60His complex, as in (F 1 -IF 1 ) 2 , the overall conformation of the ␤ DP -␣ DP catalytic interface resembles the ␤ TP -␣ TP catalytic interface in the reference structure (14,15). Several pieces of evidence indicate that in the active enzyme, ATP hydrolysis takes place at the ␤ DP catalytic site and not the ␤ TP catalytic site (6,17).…”

“…In the various structures of F 1 -ATPase, the side chain of ␣Arg-373 adopts either an ''inward pointing'' position or an ''outward pointing'' position. In both the ␤ DP and ␤ TP catalytic sites of the reference structure, of the ADP-azide inhibited enzyme (6,14,15), and of ground-state and transition-state analog structures formed with metallo-fluorides (17)(18)(19), ␣Arg-373 points in toward the catalytic site with its guanidinium group interacting with the ␤-and ␥-phosphates (or their ␥-phosphate analogs, BeF 3 Ϫ or AlF 4 Ϫ , or with azide). This position is consistent with its proposed role in stabilizing the pentavalent intermediate in ATP hydrolysis (15).…”

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F 1 –ATPase from bovine heart mitochondria

“…In many respects, the structure of F 1 -PH shown in Fig. 1 of F 1 -PH and the ground-state structure of bovine F 1 -ATPase (2JDI) (2). The α TP -, α DP -, and β TP -subunits are essentially identical in the two structures; the rmsd values for the structures are 2.54 and 1.38 Å in comparisons made with and without the central stalk, respectively.…”