Rotary protein motors

Scientific Reports

The EMBO Journal, 1998

The mechanism by which ion-flux through the membrane-bound motor module (F 0) induces rotational torque, driving the rotation of the γ subunit, was probed with a Na ⍣-translocating hybrid ATP synthase. The ATP-dependent occlusion of 1 22 Na ⍣ per ATP synthase persisted after modification of the c subunit ring with dicyclohexylcarbodiimide (DCCD), when 22 Na ⍣ was added first and ATP second, but not if the order of addition was reversed. These results support the model of ATP-driven rotation of the c subunit oligomer (rotor) versus subunit a (stator) that stops when either a 22 Na ⍣-loaded or a DCCD-modified rotor subunit reaches the Na ⍣-impermeable stator. The ATP synthase with a Na ⍣-permeable stator catalyzed 22 Na ⍣ out /Na ⍣ in-exchange after reconstitution into proteoliposomes, which was not significantly affected by DCCD modification of the c subunit oligomer, but was abolished by the additional presence of ATP or by a membrane potential (∆Ψ) of 90 mV. We propose that in the idling mode of the motor, Na ⍣ ions are shuttled across the membrane by limited back and forth movements of the rotor against the stator. This motional flexibility is arrested if either ATP or ∆Ψ induces the switch from idling into a directed rotation. The Propionigenium modestum ATP synthase catalyzed ATP formation with ∆Ψ of 60-125 mV but not with ∆pNa ⍣ of 195 mV. These results demonstrate that electric forces are essential for ATP synthesis and lead to a new concept of rotary-torque generation in the ATP synthase motor.

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1998

A model is presented in which ion translocation through the F 0 part of the ATP synthase drives the rotation of the ring of c subunits (rotor) versus the a subunit (stator). The coupling ion binding sites on the rotor are accessible from the cytoplasm of a bacterial cell except for the c subunit at the interface to the stator. Here, the binding site is accessible from the periplasm through a channel formed by subunit a. In the ATP synthesis mode, a coupling ion is anticipated to pass through the stator channel into the binding site of the adjacent rotor subunit, following the electrical potential. Occupation of this site triggers, probably by electrostatic forces, the rotation of the ring. This makes the binding site accessible to the cytoplasm, where the coupling ion dissociates. Simultaneously, this rotation moves again an empty rotor subunit into the contact site with the stator, where its binding site becomes loaded and rotation continues.

2013

The chemomechanical coupling scheme of the rotary motor V 1 -ATPase is incompletely understood. Results: Enterococcus hirae V 1 -ATPase (EhV 1 ) showed 120°steps of rotation without substeps, as commonly seen with F 1 -ATPase. Conclusion: The basic properties of rotary dynamics of EhV 1 are similar to those of Thermus thermophilus V 1 -ATPase. Significance: This study revealed the common properties of V 1 -ATPases as rotary molecular motors, distinct from those of F 1 -ATPases.

Proceedings of the National Academy of Sciences, 2001

The F 1 F 0 ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the γ and ɛ subunits in the F 1 part and c subunit ring in the F 0 part, rotates relative to a stator composed of α 3 β 3 δab 2 during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the ɛ subunit plays a key role by acting as a switch of this motor. Two different arrangements of the ɛ subunit have been visualized recently. The first has been observed in beef heart mitochondrial F 1 -ATPase where the C-terminal portion is arranged as a two-α-helix hairpin structure that extends away from the α 3 β 3 region, and toward the position of the c subunit ring in the intact F 1 F 0 . The second arrangement was observed in a structure determination of a complex of the γ and ɛ subunits of the Escherichia coli F 1 -ATPase. In this, the two C-terminal helices are apart and extend along the γ to interact with the α and ...

Proceedings of the National Academy of Sciences, 2011

Proceedings of the National Academy of Sciences, 1999

PLoS ONE, 2013

During ATP hydrolysis by F 1 -ATPase subunit c rotates in a hydrophobic bearing, formed by the N-terminal ends of the stator subunits (ab) 3 . If the penultimate residue at the a-helical C-terminal end of subunit c is artificially cross-linked (via an engineered disulfide bridge) with the bearing, the rotary function of F 1 persists. This observation has been tentatively interpreted by the unfolding of the a-helix and swiveling rotation in some dihedral angles between lower residues. Here, we screened the domain between rotor and bearing where an artificial disulfide bridge did not impair the rotary ATPase activity. We newly engineered three mutants with double cysteines farther away from the C-terminus of subunit c, while the results of three further mutants were published before. We found ATPase and rotary activity for mutants with cross-links in the single a-helical, C-terminal portion of subunit c (from c285 to c276 in E. coli), and virtually no activity when the cross-link was placed farther down, where the C-terminal a-helix meets its N-terminal counterpart to form a supposedly stable coiled coil. In conclusion, only the C-terminal singular a-helix is prone to unwinding and can form a swivel joint, whereas the coiled coil portion seems to resist the enzyme's torque.

Journal of Biological Chemistry, 2014

Background: Torque generation is important for the energy conversion of rotary ATPases. Results: Enterococcus hirae V-ATPase (EhV o V 1 ) generated larger torque than isolated EhV 1 . Conclusion: Rotor-stator interactions in EhV o V 1 are stabilized by the two peripheral stalks to generate larger torque compared with EhV 1 . Significance: Torques generated by intact V-ATPase and isolated V 1 moiety have been compared quantitatively for the first time.

Biophysical Journal, 2006

F 1-ATPase is an ATP-driven rotary molecular motor in which the central g-subunit rotates inside a stator cylinder made of a 3 b 3 subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the g-subunit at its carboxyl terminus, which forms an a helix that penetrates deeply into the stator cylinder. We used an a 3 b 3 g 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-mm beads to the g-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 ;50% reduction in torque and ;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 g-subunit are dispensable.