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

Ryohei Chiwata,Ayako Kohori,Tomonari Kawakami,Katsuyuki Shiroguchi,Shou Furuike,Kengo Adachi,Kazuo Sutoh,Masasuke Yoshida,Kazuhiko Kinosita

doi:10.1016/j.bpj.2014.04.013

Ryohei Chiwata, Ayako Kohori + Show 7 more

Open Access

https://doi.org/10.1016/j.bpj.2014.04.013

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Journal: Biophysical Journal	Publication Date: May 1, 2014
Citations: 19	License type: cc-by

Affiliation: Waseda University, Kyoto Sangyo University

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.

Highlights

F1-ATPase is a rotary molecular motor in which the central g subunit rotates inside a stator cylinder made of alternately arranged a3b3 subunits [1,2,3,4,5,6,7]
The g-dictated catalysis is best illustrated by the experiments where reverse rotation of g led to ATP synthesis, i.e., reversal of the hydrolysis reaction in the catalytic sites [14,15]
We started with a mutated thermophilic F1-ATPase (TF1) subcomplex a(C193S)3b(His10 at N-terminus)3g(S109C, I212C) that we regard as the WT for rotation and catalysis assays, and removed residues g20–249 to see if there are indispensable residues in the middle part of the rotor

Summary

Introduction

F1-ATPase is a rotary molecular motor in which the central g subunit rotates inside a stator cylinder made of alternately arranged a3b3 subunits [1,2,3,4,5,6,7]. The torque that drives g derives from ATP hydrolysis in the three catalytic sites residing at b-a interfaces, primarily hosted by a b subunit [8]. Mostly of mitochondrial F1 or MF1 [7,8,9], the b subunit binding a nucleotide and an empty b adopt quite different conformations, whereas variations in a structures are less conspicuous, leading to the proposal that nucleotide-dependent bending and unbending of b confers torque to g [10]. The g-dictated catalysis is best illustrated by the experiments where reverse rotation of g led to ATP synthesis, i.e., reversal of the hydrolysis reaction in the catalytic sites [14,15]. The g rotation and the control of catalysis must both be mediated by rotorstator interactions, which would involve specific contacts between key residues such as the hydrogen-bonding ‘‘catch’’ interactions [8]

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