None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation

Abstract

F1-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor's high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α3β3γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α3β3 subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F1, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F1 is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.

Rotation of artificial rotor axles in rotary molecular motors

“…In these constructs, only one of three FliJ helices occupies the central pore of the α 3 β 3 ring, whereas the remaining two helices are outside the cavity. The FliJ helices outside the cavity might not affect the rotation properties, as demonstrated by Chiwata et al (24). The linker sequence that connects the protrusion domainless γ with β was exactly the same as the peptide sequence used in ref.…”

“…The linker sequence that connects the protrusion domainless γ with β was exactly the same as the peptide sequence used in ref. 24. To investigate the role of the C-terminal sequence of γ in the force transmission, we prepared a series of FliJ-γ fusion constructs; 0, 9, 17, and 21 residues from the C terminus of FliJ were substituted with the equivalent number of residues from the C terminus of γ [ChF 1 (0γ), ChF 1 (9γ), ChF 1 (17γ), and ChF 1 (21γ) as shown in Fig.…”

“…Surprisingly, all of the F 1 constructs tested with engineered stator-rotor interfaces showed unidirectional rotation, suggesting that none of the γ residues is essential for torque transmission (24). These findings suggest that all rotor-constituting residues are equally responsible for torque transmission via individual specific interactions with the stator ring.…”

“…To clarify the molecular basis for the efficient chemomechanical coupling of F 1 , the stator-rotor interface has been extensively studied by truncation of the γ helices or extensive amino acid substitution of contact loop of β (23)(24)(25). Surprisingly, all of the F 1 constructs tested with engineered stator-rotor interfaces showed unidirectional rotation, suggesting that none of the γ residues is essential for torque transmission (24).…”

“…Here, we generated a V 1 -FliJ chimera (ChV 1 ) in which the FliJ sequence was fused to the C-terminal tip of the unstructured region of subunit D to enhance the stability of the complex. To forcibly hold the xenogeneic rotor in the stator ring, we assessed F 1 -FliJ chimeras (ChF 1 ) by genetically fusing the β subunit of F 1 to FliJ using a flexible linker as previously described (24). We found both chimeric motors function as active rotary motors.…”