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

Matthew W Bowler,Martin G Montgomery,Andrew G.W Leslie,John E Walker

doi:10.1074/jbc.m700203200

Matthew W Bowler, Martin G Montgomery + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m700203200

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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 1.9A resolution. This structure has been compared with the previously described structure of bovine F(1)-ATPase determined at 1.95A 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 beta(DP)-subunit. In the present structure, the nucleotide binding sites in the beta(DP)- and beta(TP)-subunits are both occupied by AMP-PNP, whereas in the earlier structure, the beta(TP) site was occupied by AMP-PNP and the beta(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, alphaArg-373 differs in the beta(DP)- and beta(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.

Highlights

14238 JOURNAL OF BIOLOGICAL CHEMISTRY tions with different nucleotide occupancies
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 F1-ATPase [2], it was proposed that the interconversion of sites is effected by a mechanical rotation of the ␥-subunit, each 360° rotation taking each ␤-subunit through the three states and thereby hydrolyzing three ATP molecules
In a related structure at 1.95 Å resolution, determined with crystals grown under conditions identical to those used in the first structural analysis, azide was resolved in the ␤DP-subunit [6]

Summary

Ground State Mitochondria

In a related structure at 1.95 Å resolution, determined with crystals grown under conditions identical to those used in the first structural analysis, azide was resolved in the ␤DP-subunit [6] It is associated via the partial positive charge of its central nitrogen atom with the ␤-phosphate of ADP and via the partial negative charges of its two peripheral nitrogen atoms with ␤Lys-162 in the P-loop region of the nucleotide binding site and with the catalytically essential residue, ␣Arg-373. Crystals of bovine F1-ATPase were grown in the presence of both ADP and AMP-PNP under conditions identical to those employed previously, except that azide was omitted The structure of this complex determined at 1.9 Å resolution (see footnote b in Table 1) shows that, despite the presence of both ADP and AMP-PNP, both the ␤TP and ␤DP nucleotide binding sites were occupied by AMP-PNP. Azide-free F1-ATPase tation to date of an intermediate in the catalytic cycle of ATP hydrolysis by the enzyme

EXPERIMENTAL PROCEDURES

Space group

RESULTS AND DISCUSSION