Neither Helix in the Coiled Coil Region of the Axle of F₁-ATPase Plays a Significant Role in Torque Production

Mohammad Delawar Hossain ^* , Shou Furuike ^*, Yasushi Maki , Kengo Adachi ^*, Toshiharu Suzuki  ^¶, Ayako Kohori ^*, Hiroyasu Itoh ^|| **, Masasuke Yoshida  ^¶ and Kazuhiko Kinosita, Jr. ^*

^* Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-Ku, Tokyo 169-8555, Japan; Department of Physics, School of Physical Sciences, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh; Department of Physics, Osaka Medical College, Osaka 569-8686, Japan; Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503, Japan; ^¶ ATP-Synthesis Regulation Project, International Cooperative Research Project, Japan Science and Technology Agency, Tokyo 135-0064, Japan; ^|| Tsukuba Research Laboratory, Hamamatsu Photonics KK, Tokodai, Tsukuba 300-2635, Japan; and ^** Team 13, Formation of Soft Nano-Machines, Core Research for Evolutional Science and Technology, Tokodai, Tsukuba 300-2635, Japan

Correspondence: Address reprint requests to Kazuhiko Kinosita Jr., Dept. of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-Ku, Tokyo 169-8555, Japan. Tel.: 81-3-5952-5871; Fax: 81-3-5952-5877; E-mail: kazuhiko_at_waseda.jp

F₁-ATPase is an ATP-driven rotary molecular motor in which the central -subunit rotates inside the cylinder made of ₃β₃ subunits. The amino and carboxy termini of the -subunit form the axle, an -helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation. Here we examine the role of each helix by truncating only the carboxy terminus by 25–40 amino-acid residues. Longer truncation impaired the stability of the motor complex severely: 40 deletions failed to yield rotating the complex. Up to 36 deletions, however, the mutants produced an apparent torque at nearly half of the wild-type torque, independent of truncation length. Time-averaged rotary speeds were low because of load-dependent stumbling at 120° intervals, even with saturating ATP. Comparison with our previous work indicates that half the normal torque is produced at the orifice of the stator. The very tip of the carboxy terminus adds the other half, whereas neither helix in the middle of the axle contributes much to torque generation and the rapid progress of catalysis. None of the residues of the entire axle played a specific decisive role in rotation.