Torque Generation in F1-ATPase Devoid of the Entire Amino-Terminal Helix of the Rotor That Fills Half of the Stator Orifice

Abstract

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α(3)β(3) subunits. The amino and carboxyl termini of the γ rotor form a coiled coil of α-helices that penetrates the stator cylinder to serve as an axle. Crystal structures indicate that the axle is supported by the stator at two positions, at the orifice and by the hydrophobic sleeve surrounding the axle tip. The sleeve contacts are almost exclusively to the longer carboxyl-terminal helix, whereas nearly half the orifice contacts are to the amino-terminal helix. Here, we truncated the amino-terminal helix stepwise up to 50 residues, removing one half of the axle all the way up and far beyond the orifice. The half-sliced axle still rotated with an unloaded speed a quarter of the wild-type speed, with torque nearly half the wild-type torque. The truncations were made in a construct where the rotor tip was connected to a β-subunit via a short peptide linker. Linking alone did not change the rotational characteristics significantly. These and previous results show that nearly half the normal torque is generated if rotor-stator interactions either at the orifice or at the sleeve are preserved, suggesting that the make of the motor is quite robust.

Robustness of the Rotary Catalysis Mechanism of F1-ATPase

“…The stiffness of the rotary potential is a good barometer of how strongly the ␣␤ stator ring holds the ␥ subunit. This correlation was also observed in a previous study of a mutant with extensive alanine substitution at the DELSEED loop of the ␤ subunit (44,47,48). Results of this study also indicated that the P-loop lysine, general base, and arginine finger all play a role to tighten the interaction between ␤ and ␥, which likely stabilizes the closed conformation of the ␤ subunit.…”

Biophysical Characterization of a Thermoalkaliphilic Molecular Motor with a High Stepping Torque Gives Insight into Evolutionary ATP Synthase Adaptation

“…The lowering of K m is a known hallmark for competitive inhibition, suggesting that inactive molecules are likely due to ADP inhibition. In support of this, it is well known that ATP hydrolysis in TF 1 is regulated by Mg-ADP inhibition to prevent wasteful ATP consumption (57), a phenomenon yet to be explored in TA2F 1 (24,44,59).…”

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

“…All direct measurements (see ) for a recent review), both on V-and F-ATPase, rely on gene-engineered modification of the enzyme in order to do single molecule fluorescence resonance energy transfer experiments, or, more commonly attach a visualisation elements, e.g., fluorescent filament or polystyrene or gold bead, to the rotor (or stator) and fix the stator (or rotor) on a solid support, followed by visualisation of the rotation Tsunoda et al 2001;Nishio et al 2002;Hirata et al 2003;Nakanishi-Matsui et al 2006;Xie 2009;Sekiya et al 2010;Furuike et al 2011;Kohori et al 2011;Okuno et al 2011). Such modifications may add or remove barriers to the rotation not present in the native system.…”