Researchers from Japan have
successfully elucidated the fine details of energy-transducing F1-ATPase
function in bacteria
F-Type ATP synthase, a catalytic complex of proteins, synthesizes
adenosine triphosphate (ATP), the energy currency of living cells. A lot of
ambiguity exists over the rotational mechanism of this spinning enzyme. Now, researchers
from Japan have demonstrated how each chemical event of ATP metabolism is
linked to the ‘stepwise’ rotational movement of the F1 component of ATPase.
Especially, they clarified the angle of shaft rotation before ATP-cleavage, a
long-standing enigma, to be 200°.
Adenosine triphosphate (ATP) is the energy currency of
cells. It powers various cellular processes that require energy, including enzymatic
reactions. ATP is synthesized with the help of an enzyme complex called F-type ATP
synthase. This enzyme complex has a bidirectional functionality, working to synthesize
ATP as well as hydrolyzing it, depending on environmental and cellular cues. ATP
synthase consists of two rotating motors—F1 and F0. The F1
subcomplex is mainly composed of α, β, and γ subunits. During the hydrolysis of
ATP, the F1-ATPase show rotational motion. Therefore, F1-ATPase
is also known as the world’s smallest rotary biological molecule motor.
However, the underlying mechanism of how ATP hydrolysis makes the molecule
rotate remains unclear.
To address this knowledge gap, a team of researchers from
Japan, led by Associate Professor Tomoko Masaike from Tokyo University of
Science, set out to investigate the events behind the rotational mechanism of F1-ATPase
in the thermophilic bacterium, Bacillus PS3. Elaborating on the
objectives of their study, Dr. Masaike explains, “We wanted
to clarify the mechanism by which F1-ATPase rotates the central shaft
during ATP hydrolysis. We focused on clarifying the angle of rotation of the
central shaft between the binding of the substrate ATP to the enzyme and the
cleavage of its high-energy phosphate bond.” The study, which was
undertaken in collaboration with Professor Takayuki Nishizaka from Gakushuin
University and Yuh Hasimoto from Hamamatsu Photonics K.K., was made available
online on 21 December 2022 and 7 February 2023
in print in Biophysical Journal.
Previous investigations of the F1 subunits of Bacillus PS3 have established that ATP cleavage involves chemomechanical coupling, i.e., each rotational stepping
motion is linked to a chemical reaction step. The angle of rotation between ATP binding and its cleavage at the same
catalytic site has been previously estimated to be 200°. However, experimental
evidence to substantiate this has so far been lacking. To address this, the
researchers studied the rotation by creating a hybrid F1 using one
mutant β and two wild type βs. Since the rate of both ATP cleavage and ATP
binding was extremely slow in the mutant, the researchers could observe the pauses
or dwells between rotational steps easily.
Upon performing a single-molecule rotation assay with varying
concentrations of ATP, they could observe three sets of short and long dwells associated
alternately with 80° and 40° intervals per revolution. To
investigate the events associated with the dwells, the authors performed
dwell-time analyses. The long pause before the 40° sub-step was independent of ATP
concentration and was confirmed as the ‘catalytic dwell’—a pause in the rotation
of the shaft due to ATP cleavage. Alternately, the short pause before the 80° step was clearly dependent on ATP
concentration and thus identified as the ‘ATP-waiting dwell’ (pause to enable β subunit to bind ATP). “Upon
investigating the rotation of the shaft, we could provide visible evidence
through optical microscopy that the shaft angles at ATP-binding and cleaving
events in Bacillus PS3 were 0° and 200°, respectively” says Hasimoto.
With this study, the authors have resolved a long-term
debate over the ATP-cleavage shaft angle and established the chemomechanical
correlation of ATPase function. Talking about the future impacts of their novel
study, Associate Prof. Masaike elaborates,
“Since F1-ATPase is the
world’s smallest biological rotational motor protein, it can be used as a
reference to understand the mechanism of energy transduction in living
organisms. This knowledge can be revolutionary in developing efficient
nanomachines. Moreover, ATP synthase from Mycobacterium tuberculosis
has recently been identified as a potential target for drug discovery.
Therefore, to stop its rotation using inhibitors, understanding the mechanism of
rotation is quite important.”
Indeed, understanding the world’s smallest biological
motor may unravel mysteries of energy transduction in living organisms, and may
even translate to advanced applications across disciplines!
***
Reference
Title of original paper:
Journal: Biophysical Journal
DOI: https://doi.org/10.1016/j.bpj.2022.12.027
Authors: Yuh Hasimoto1,*,
Mitsuhiro Sugawa2, Yoshihiro Nishiguchi3, Fumihiro Aeba4,
Ayari Tagawa4, Kenta Suga4, Nobukiyo Tanaka4,
Hiroshi Ueno5, Hiroki Yamashita4, Ryuichi Yokota4,
Tomoko Masaike4,*, and Takayuki Nishizaka3,*
Affiliations:
1Tsukuba
Research Center, Central Research Laboratory, Hamamatsu Photonics K.K.
2Graduate
School of Arts & Sciences, The University of Tokyo
3Department
of Physics, Faculty of Science, Gakushuin University
4Department
of Applied Biological Science, Faculty of Science and Technology, Tokyo
University of Science
5Department
of Applied Chemistry, Graduate School of Engineering, The University of Tokyo
*Corresponding
authors
Funding information
This study was supported in part by a
Grant-in-Aid for Scientific Research on Innovative Areas [“Fluctuation &
Structure” of JP16H00808 and JP26103527 (to T.N.), “Cilia & Centrosomes” of
JP87003306 (to T.N.)], Grant-in-Aid for Research Activity Start-up JP22870028
(to T. M.), and PRESTO JPMJPR12L8, JST (to T. M.).
Further Information
Associate Professor Tomoko Masaike
Department of Applied Biological
Science
Tokyo University of Science
Email: [email protected]
Professor Takayuki Nishizaka
Department of Physics
Gakushuin University
Email: [email protected]
Media contact
Hiroshi Matsuda
Public Relations Division
Tokyo University of Science
Email: [email protected]
Website: https://www.tus.ac.jp/en/mediarelations/
Ayano Kawasaki
President’s Office Public Relations Centre
Gakushuin University
Email: [email protected]
