The rotor tip inside a bearing of a thermophilic F1-ATPase is dispensable for torque generation.
F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside a stator cylinder made of alpha(3)beta(3) subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the gamma-subunit at its carboxyl terminus, which forms an alpha helix that penetrates deeply into the stator cylinder. We used an alpha(3)beta(3)gamma subcomplex of F(1)-ATPase derived from thermophilic Bacillus PS3 and expressed it in Escherichia coli. We could obtain purified subcomplexes in which 14, 17, or 21 amino-acid residues were deleted. The rotary characteristics of the truncated mutants, monitored by attaching a duplex of 0.49-microm beads to the gamma-subunit, did not differ greatly from those of the wild-type over the ATP concentrations of 20 nM-2 mM, the most conspicuous effect being approximately 50% reduction in torque and approximately 70% reduction in the rate of ATP binding upon deletion of 21 residues. The ATP hydrolysis activity estimated in bulk samples was more seriously affected. The 21-deletion mutant, in particular, was >10-fold less active, but this is likely due to instability of this subcomplex. For torque generation, though not for rapid catalysis, most of the rotor-stator contacts on the deeper half of the penetrating portion of the gamma-subunit are dispensable.
Hossain
Mohammad Delawar
MD
Department of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Furuike
Shou
S
Maki
Yasushi
Y
Adachi
Kengo
K
Ali
M Yusuf
MY
Huq
Mominul
M
Itoh
Hiroyasu
H
Yoshida
Masasuke
M
Kinosita
Kazuhiko
K
D001407:Bacillus / Q000737:chemistry; D020134:Catalytic Domain; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D019415:Torque
D020409:Molecular Motor Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases