Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation.
F(1)-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the gamma-subunit against the alpha(3)beta(3) ring. The crystal structures of F(1), which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F(1) has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F(1), it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at betaE391 and gammaR84 of F(1) from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F(1), the corresponding residues in the ADP-bound beta (beta(DP)) and gamma were in direct contact. The betaE190D mutation was additionally introduced into the beta to slow ATP hydrolysis. By incorporating a single copy of the mutant beta-subunit, the chimera F(1), alpha(3)beta(2)beta(E190D/E391C)gamma(R84C), was prepared. In single-molecule rotation assay, chimera F(1) showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of beta(E190D/E391C). When the mutant beta and gamma were cross-linked through a disulfide bond between betaE391C and gammaR84C, F(1) paused the rotation at the catalytic dwell angle of beta(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that beta(DP) is the catalytically active form. The former point was again confirmed in experiments where F(1) rotation was inhibited by adenosine-5'-(beta,gamma-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F(1).
Okuno
Daichi
D
The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
Fujisawa
Ryo
R
Iino
Ryota
R
Hirono-Hara
Yoko
Y
Imamura
Hiromi
H
Noji
Hiroyuki
H
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D009154:Mutation; D020836:Protein Structure, Quaternary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases