Challis, Katharine J.

Jack, Michael W.

Elsevier - Publisher Connector 

A theoretical treatment of energy transfer in a molecular motor based on Brownian motion on a multidimensional free-energy landscape: The derivation of formal analytic results in the Kramers regime of deep potential wells 

Esparza-Perusquía is a PhD student of the Ciencias Biológicas
(511021118) of Universidad Nacional Autónoma de México and
fellow to CONACYT (254400).
doi:10.1016/j.bbabio.2014.05.199
S1.P7
The role of residue beta249 in MgADP inhibition of
Escherichia coli ATP synthase
Boris Feniouka, Anastasia Prikhodkoa, Anna Lapashinab,
Maria Vitushkinac, Roman Zinovkinc
aFaculty of Bioengineering and Bioinformatics, Lomonosov Moscow
State University, Russia
bLomonosov Moscow State University, Moscow, Russia
cFaculty of Biology, Lomonosov Moscow State University, Russia
E-mail address: boris.a.feniouk@gmail.com
When MgADP without phosphate is bound at a catalytic site of
H+-FoF1 ATP synthase, there is a probability that the enzyme gets
blocked in an inactive conformation. This phenomenon is known as
MgADP inhibition. Earlier experiments indicate that the enzyme
adopts a special inactive conformation with MgADP trapped in a
catalytic site. This is a core feature of ATP synthase observed even in
the minimal catalytic alpha3beta3gamma-complex. MgADP inhibi-
tion is found in all ATP synthases studied so far. However, the
degree of inhibition varies considerably, being strong in mitochon-
dria and especially chloroplast enzyme, as well as in some bacteria,
e.g. Bacillus PS3, but rather weak in FoF1 from Escherichia coli. We
have demonstrated previously that in Bacillus PS3 ATP synthase
mutation betaGln259Leu can dramatically decrease MgADP inhibi-
tion. In E. coli wild type enzyme the residue in the corresponding
position (beta249) is Leu, i.e. it is similar to the mutant Bacillus PS3
enzyme. In this work we made an E. coli mutant betaLeu249Gln. The
mutation signiﬁcantly enhanced MgADP inhibition. Moreover, it
completely changed the effect of phosphate on MgADP inhibition,
while in the wild-type enzyme phosphate increased the inhibitory
effect of MgADP, and in the betaLeu249Gln it relieved the inhibition.
We conclude that MgADP inhibition is a relatively easily modulated
regulatory feature of ATP synthase rather than an inevitable “side
effect” of the rotary catalytic mechanism.
doi:10.1016/j.bbabio.2014.05.200
S1.P8
The Fo complex of the chloroplast ATP synthase — Towards a
structural study of a macromolecular nanomotor
Alexander Hahn, Thomas Meier
Max Planck Institute of Biophysics, Germany
E-mail address: alexander.hahn@biophys.mpg.de
Powered by the proton-motive force (pmf) of the cell, F-type ATP
synthases are the major producers of adenosine triphosphate (ATP)
in cells. The ATP synthase in plant chloroplasts consists of two
domains, the soluble F1 and the membrane-embedded Fo sub-
complex. In Fo the pmf across the thylakoid membrane is converted
into torque and induces rotation of a rotor element, the c-ring. The
unique construction of the Fo complex allows ions (H+ or Na+) to
pass a distinct pathway through the rotor–stator elements and
surmount the membrane from one side to another. The rotation is
tightly coupled with F1 and elicits conformational changes in the
catalytic beta-subunits, ﬁnally leading to ATP synthesis. While the
function of the F1 complex and the Fo rotor element has unveiled by
many high-resolution structures in the last 20 years, structural informa-
tion about the complete Fo complex or its neighboring stator subunits a
and b is still scarce. To understand the molecular mechanism of this
nanomotor, high resolution structural information is indispensable. We
set out to investigate the chloroplast Fo-ATP synthase complex by
structural methods. By combining classical biochemical puriﬁcation
strategies such as sucrose density centrifugation and ion-exchange
chromatography we were able to establish a protocol for a large scale
puriﬁcation of the ac14 sub-complex of the Spinacia oleracea ATP-
synthase. The isolation of abundant and stable ac14 allows subsequent
experiments such as 2D and 3D crystallization and their structural
characterization by electron microscopy and X-ray crystallography.
doi:10.1016/j.bbabio.2014.05.201
S1.P9
A theoretical treatment of energy transfer in amolecularmotor based
on Brownian motion on a multidimensional free-energy landscape:
The derivation of formal analytic results in the Kramers regime of
deep potential wells
Michael W. Jack, Katharine J. Challis
Scion, New Zealand
E-mail address:michael.jack@scionresearch.com
In a cell, specialised proteins act as molecular-scale motors to
convert energy from one form to another. For example, ATP synthase
converts chemical energy into mechanical work and back again, ion
pumps convert chemical energy into transport against an electrochem-
ical gradient, and cytoskeletal motors use chemical energy to transport
loads along linear molecular chains. Despite functional differences
between different types of molecular motors, there are fundamental
similarities in their behaviour and properties: i) they transfer energy
between different degrees of freedom (i.e., chemical and mechanical),
ii) they are subject to large thermal ﬂuctuations, and iii) they operate far
from equilibrium.We present a general theoretical treatment of energy
transfer in a molecular motor based on Brownian motion on a
multidimensional free-energy landscape. This treatment enables ener-
gy transfer between degrees of freedom to be described explicitly and
captures key fundamental elements of molecular motor operation.
Unfortunately, the multidimensional diffusion equation arising in this
treatment is not solvable in general. We transform the continuous
diffusion equation to a simpler discrete master equation that is
analytically tractable [1]. This provides an opportunity to connect the
general continuous multidimensional Brownian-motion based theory
with experiments, phenomenological models, and established results
from non-equilibrium thermodynamics. In the Kramers regime of deep
potential wells, we derive a range of formal expressions for molecular
motors. We determine physical properties including the drift and
diffusion, the rate and efﬁciency of energy transfer, and the entropy
generated [2]. We also consider thermal ﬂuctuations for the motor and
show that energy transfer between degrees of freedom creates
statistically correlated ﬂuctuations in those degrees of freedom [3].
The ﬂuctuation statistics provide an opportunity to distinguish different
operating regimes of the motor.
References
[1] K. J. Challis and M. W. Jack, Tight-binding approach to overdamped
Brownian motion on a multidimensional tilted periodic potential,
Physical Review E 87, (2013) 052102.
[2] K. J. Challis and M. W. Jack, Energy transfer in a molecular motor in
the Kramers regime, Physical Review E 88, (2013) 042114.
Abstracts e15
[3] K. J. Challis and M. W. Jack, Thermal ﬂuctuation statistics in a
molecularmotor described by amultidimensionalmaster equation,
Physical Review E 88, (2013) 062136.
doi:10.1016/j.bbabio.2014.05.202
S1.P10
The ingenious structure of central rotor apparatus in VoV1: Key for
both complex disassembly and energy coupling between V1 and Vo
Jun-ichi Kishikawaa, Atsuko Nakanishia,
Masatada Tamakoshib, Ken Yokoyamaa
aKyoto Sangyo University, Japan
bTokyo University of Pharmacy and Life Science, Japan
E-mail address: kishi.jun@cc.kyoto-su.ac.jp
The vacuole-type ATPases (VoV1) are found in many organisms
and are involved in a variety of physiological processes. The VoV1 and
FoF1 ATPases/synthases are evolutionarily related and share a rotary
mechanism to perform their speciﬁc functions. The basic structures
of the ATPases/synthases are conserved among species. The soluble,
cytoplasmic portion of FoF1 and VoV1 (called F1 and V1, respectively),
responsible for ATP hydrolysis/synthesis, is connected via the central
shaft and the peripheral stator stalk to the transmembrane portion
(Fo and Vo) that houses the ion transporting pathway. In FoF1, a
central shaft, γ-subunit, binds directly to the rotor ring. In contrast,
at the boundary surface of VoV1, Vo-C forms a socket-like structure
which accommodates the V1-DF central shaft, indicating that V1-DF
does not contact the rotor ring directly. Thus, the boundary surface
of VoV1 is signiﬁcantly different from that of FoF1. In this study,
reconstitution and ﬂuorescence resonance energy transfer analysis of
VoV1 subcomplexes revealed a weak binding afﬁnity of V1-DF to Vo-C
despite the fact that torque needs to be transmitted between V1-DF
and Vo-C for the tight energy coupling between V1 and Vo. Mutation
of a short helix at the tip of V1-DF caused intramolecular uncoupling
of VoV1 and a decrease in binding afﬁnity of the mutated V1 for Vo,
suggesting that proper ﬁtting of the short helix of V1-D into the
socket of Vo-C is required for tight energy coupling between V1 and
Vo. To account for the apparently contradictory properties of the
interaction between V1-DF and Vo-C (weak binding afﬁnity but
strict requirement for torque transmission), we propose a model in
which the relationship between V1-DF and Vo-C corresponds to that
between a slotted screwdriver and a minus thread screw. This model
is consisted of our previous result in which the central rotor
apparatus is not the major factor for the association of V1 with Vo.
doi:10.1016/j.bbabio.2014.05.203
S1.P11
Molecular basis of ADP-inhibition of V type ATPase/synthase
Jun-ichi Kishikawa, Atsuko Nakanishi
Kyoto Sangyo University, Japan
E-mail address: kishi.jun0724@gmail.com
Vacuolar type ATPase (VoV1) functions as an ATP hydrolysis-driven
proton pump that carries out acidiﬁcation of cellular compartments in
eukaryotes. A family of VoV1, sometimes referred to as the A-type
ATPases or AoA1, is also found in archaea and some eubacteria
(the prokaryotic VoV1 family). In most prokaryotes, such as Thermus
thermophilus, the VoV1 functions as an ATP synthase. However it can
also act as a primary ion pump, as seen in Enterococcus hirae.
ADP-inhibition caused by entrapment of ADP at a catalytic site is
believed to be a regulatory mechanism of FoF1 to prevent wasteful ATP
consumption when proton motive force is lost. VoV1 from T. thermophilus
(TthVoV1) also exhibits sensitivity to ADP inhibition, resulting in rapid
decay of the ATPase activity of the V1 subcomplex. Contrary, VoV1 of
E. hiraeor eukaryotes showno sensitivity toADP-inhibition, relevant to the
physiological role of this ion pump. ADP-inhibition has been investigated
in FoF1, but the precise molecular mechanism remains poorly understood.
To investigate the molecular basis for this difference, domain
swapped chimeric V1 consisting of both T. thermophilus and E. hirae
enzymes was generated and its function analyzed. The data showed
that the interaction between the nucleotide binding and C terminal
domains of the catalytic A subunit from E. hirae V1 is central to
increasing binding afﬁnity of the chimeric V1 for phosphate, resulting in
reduction of the ADP-inhibition. These ﬁndings together with a
comparison of the crystal structures of T. thermophilus V1 with E. hirae
V1 strongly suggest that the A subunit adopts a different conformation
in T. thermophilus V1 from that in E. hirae V1. This key difference results
in ADP inhibition of T. thermophilusV1 by abolishing the binding afﬁnity
for phosphate during ATP hydrolysis.
doi:10.1016/j.bbabio.2014.05.204
S1.P12
The effect of betaq259l mutation onMgADP-inhibition of B. subtilis
H+-FOF1-ATP-synthase
Anna Lapashinaa, Maria Vitushkinab, Roman Zinovkinb, Boris Fenioukc
aLomonosov Moscow State University, Moscow, Russia
bFaculty of Biology, Lomonosov Moscow State University, Russia
cFaculty of Bioengineering and Bioinformatics, Lomonosov Moscow State
University, Russia
E-mail address: a.lapashina@gmail.com
The ATPase activity of H+-FOF1-ATP synthase can be regulated in
several ways, but the most common of them is inhibition by MgADP. It
is known for all studied FOF1 complexes thatMgADPbound in a catalytic
site in the absence of phosphatemay cause enzyme inactivation instead
of being released and replaced by new MgATP. The degree of this
inhibition varies greatly between different organisms: the inhibition is
strong in chloroplast FOF1 and enzymes of some bacteria (Bacillus PS3,
Bacillus subtilis), but in FOF1 of Escherichia coli it is much weaker.
It has been reported previously [1] that a point mutation
Gln259Leu in the beta subunit of Bacillus PS3 FOF1 noticeably relieves
its strong MgADP inhibition. In this work, we inserted the same
mutation in B. subtilis. MgADP inhibition in our mutant FOF1 also
weakened signiﬁcantly in comparison to the wild-type enzyme. The
mutant was capable of ATP-driven proton pumping and was sensitive
to azide like the wild type. The initial ATPase activity levels of the
mutant and of the wild-type FOF1 complexes were comparable
at room temperature and at 37 °C, but the mutant enzyme has
demonstrated sensitivity to cold temperatures. The living cells of the
mutant strain also were not resistant to cold. To clarify the
physiological role of MgADP inhibition in B. subtilis, we also compared
wild-type and mutant strains in competition growth experiments
under different conditions.
Reference
[1] B. A. Feniouk, C. Wakabayashi, T. Suzuki, M. Yoshida, A point
mutation, betaGln259Leu, relieves MgADP inhibition in Bacillus
PS3 ATP synthase, BBA - Bioenergetics, 1817 (2012) S22.
doi:10.1016/j.bbabio.2014.05.205
Abstractse16


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A theoretical treatment of energy transfer in a molecular motor based on Brownian motion on a multidimensional free-energy landscape: The derivation of formal analytic results in the Kramers regime of deep potential wells

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