Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 52, Pages 20722-20727Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0805828106
Keywords
ATP synthase; cross-link
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Funding
- Ministry of Education, Culture, Sports, Science, and Technology of Japan [18074005]
- Grants-in-Aid for Scientific Research [18074005] Funding Source: KAKEN
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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 beta E391 and gamma R84 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 beta E190D 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 beta E391C and gamma R84C, 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.
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