pISSN 2288-6982 l eISSN 2288-7105
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MINI REVIEW P 1-11
Structural insights on ATP hydrolysis-driven
mechanical work of AAA+ hexamers
Changwon Kim1,2,4, Sang-Hyun Rah1,2,3,4 and Tae-Young Yoon1,2,4*
1
Center for Nanomedicine, Institute for Basic Science (IBS), Yonsei University, Seoul 03722, Korea, 2Yonsei-IBS Institute, Yonsei
University, Seoul 03722, Korea, 3Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141,
South Korea, 4present address : School of Biological Sciences, Seoul National University, Seoul 08826, Korea
*Correspondence: [email protected]
N-ethylmaleimide-sensitive factor (NSF) and ClpX are homo-hexameric proteins of the AAA+ (ATPases Associated with
diverse cellular Activities) family. Using ATP, NSF recycles SNARE complexes following membrane fusion, while ClpX
unfolds and translocates proteins through its pore. However, their molecular mechanisms were unclear until recently. NSF
efficiently disassembles a SNARE complex using ATP that were bound before SNARE binding, by changing from a ‘split
washer’ to a ‘flat washer’ conformation. ClpX utilizes numerous ATP binding and hydrolyses for translocation. Structural
studies of ClpX show that two of the six ATP sites are unloadable. Hence, in ClpX, it is believed ATP hydrolyses occur in
pairs and in symmetric motifs to work. Overall, NSF follows a spring loaded model, while ClpX follows a power-stroke
model – showing that even for proteins that belong to the same family and that have similar structures, functions and
models of action can be very different.
INTRODUCTION
The AAA+ (ATPases Associated with diverse cellular Activities)
protein domain exists in many proteins (Hanson and Whiteheart,
2005), and performs various essential functions in the cell by
utilizing energy from ATP hydrolysis (Iyer et al., 2004; Sauer and
Baker, 2011; Ulbrich et al., 2009; Xu et al., 2009; Zhu et al., 2008).
Despite their importance, the connection between structure, ATP
hydrolysis, and function were a mystery until the development of
various structural, biochemical, and single-molecule techniques
(Aubin-Tam et al., 2011; Chang et al., 2012; Kim and Kim, 2003;
Martin et al., 2005; Min et al., 2013; Rodriguez-Aliaga et al.,
2016; Ryu et al., 2015; Stinson et al., 2015; Stinson et al., 2013;
Zhao et al., 2015). In this review, we focus on recent findings,
similarities and differences of two well studied hexameric AAA+
proteins, ClpX and NSF.
Monomers of both ClpX and NSF consist of three domains.
Both have N-terminal domains that are related to substrate
binding. The remaining domains are all AAA+ domains, which
are called D1 and D2 in NSF (Fleming et al., 1998; Hanson et
al., 1997; Lenzen et al., 1998; Yu et al., 1998), and large and
small AAA+ domains in ClpX. ClpX hexamerizes via each large
domain binding with the small domain of the subunit to its anticlockwise direction (Kim and Kim, 2003). There are hinge regions
between the large and small domain, where ATP can bind and be
hydrolyzed for enzyme activity. In contrast, NSF’s D2 domains all
bind ATP to connect the monomers, while the D1 domains are all
responsible for catalytic activity only.
The distances between the C-term of a subunit and the
adjacent subunits’ N-terms differ depending on the position (right
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or left) from the subunit. Also, if a special protein tag is used,
ClpX can recognize substrates even if its N domain is removed.
Based on such facts, functional oligomers of ∆N-ClpX subunits
connected with flexible peptide linkers could be designed. This
methodology was a powerful tool for studying the structure and
function of ClpX, as point mutations could be introduced to any
subunit of the hexamer as desired (Martin et al., 2005).
However, for NSF, such methodology cannot be applied
because the size is two times bigger than ClpX. Furthermore,
NSF was found to carry out its functions in a single burst, making
studies on ATP hydrolysis inherently more difficult. Even so,
various high–resolution structural analysis and single molecule
approaches are gradually shedding light on the two proteins’
molecular details.
CRYSTAL STRUCTURE OF CLPX AND ITS
ATP BINDING SITE
ClpX is a ring-shaped AAA+ machine that unfolds and
translocates various protein substrates through its pore. ClpX
forms a complex with ClpP, which accepts the translocated
protein and subsequently degrades it (Figure 1A). ClpX’s
N-domain is a C4-type zinc binding domain, and forms
constitutive dimers. This ZBD is responsible for recognizing most
substrates of ClpX (Wojtyra et al., 2003). So if the N-domain
of ClpX is removed, the resulting protein (∆N-ClpX) retains its
ability to bind with ClpP, but cannot recognize most substrates.
However, if special tags are used, substrate recognition is
possible even without the N-domain. An example is the ssrA
tag, which is normally attached to the C-termini of incompletely
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Structural insights on ATP hydrolysis-driven mechanical work of AAA+ hexamers
A
A
A
substrate
substrate
A
ClpX
ClpX
ClpP
unfolding
translocation
unfolding
translocation
degradation
substrate
ClpX
ClpP
unfolding
translocation
degradation
ClpP
degradation
C
B
B
B
C
small
B
large
small
N
C
C
large
N
small
hinge
small large
large
domains
C
C
large
large
domains
domains
small
large
N
N
135Å
C
N
135Å
N
small
135Å
small
hinge
large
small
domains
domains
hinge
large
unloadable
unloadable
D
D
D
D
L
L
small
small
large
large
Rigid-body
unit
U
L
L
loadable
L
L
large
82°
unloadable
small
small
82°
loadable
82° loadable
domains
U
U
Rigid-body
unit
U
L
Rigid-body
unit
L
U
U
nucleotide
L
L
Individual
subunit
FIGURE 1 I ClpX mechanism and structure. (A) Function of ClpX and ClpP. ClpX unfolds and translocates protein substrates to ClpP, and ClpP degrades the
L and a small domain,
L
substrate. (B) Cartoon of ∆N-ClpX. A subunit is formed of a large domain
connected by an ATP-binding hinge. Distance between the C-term of
a subunit and adjacent subunits’ N-terms differ depending on the direction. (PDB 3HWS) (C) Loadable and unloadable structures exist, and the angle of the small
nucleotide
domain differs largely. (D) 2-fold symmetry ∆N-ClpX structure.
Only four nucleotides are bound,
resulting in a 4L:2U structure. (C) is adapted by permission from
Individual
ELSEVIER Ltd: [Cell] (Glynn, Martin, Nager, Baker, & Sauer, 2009) copyright.
subunit
nucleotide
translated polypeptides. Once a substrate is recognized by the
ssrA tag, translocation occurs irrespective of the presence of
the N-domain. Therefore, ATPase activity of ClpX can be studied
even with ∆N-ClpX (Dougan et al., 2003; Singh et al., 2001).
How ATP hydrolysis by ClpX is transformed to mechanical
work attracted a lot of interest, especially since the crystal
structure was solved in 2003 (Kim and Kim, 2003). Although the
structure solved in this work was ∆N-ClpX, it provided essential
information to start deciphering the molecular mechanism. Each
subunit of ∆N-ClpX consisted of a large domain and a small
domain, with a nucleotide-binding hinge domain in between
(Figure 1B). (Kim and Kim, 2003). When the distance from a
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Individual
subunit
subunit’s C-term to the N-termini of the neighboring subunits
were measured, it was found that in the clockwise direction, the
distance is about 45Å, while for the anti-clockwise direction the
distance was about 95Å. Hence, a flexible polypeptide linker of
about 70 Å was used to link thee subunits (Martin et al., 2005),
and this construct was used to produce a crystal structure with
two-fold symmetry (Figure 1B) (Glynn et al., 2009). The biggest
discovery from this new structure was that only four subunits
had nucleotides bound (Figure 1D) – which was not evident in
the previous work, as a six-fold symmetry was assumed. The
two kinds of subunits with or without nucleotides bound had a
significant structural difference in the small domain (Figure 1C),
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Changwon Kim, Sang-Hyun Rah and Tae-Young Yoon
and became to be called L (loadable) subunits and U (unloadable)
subunits, respectively. This discovery became the basis for
elucidating the molecular models of ATP hydrolysis.
as concerted, sequential, probabilistic models, and possible
subunit configurations were proposed (Figure 2A) (Martin et
al., 2005). However, it was not until the application of singlemolecule methods more valid models could be selected and
refined. (Aubin-Tam et al., 2011; Rodriguez-Aliaga et al., 2016;
Stinson et al., 2013)
In a study by Stinson et. al. (Stinson et al., 2013), metal iondependent dye quenching (coordinated metal energy transfer;
CoMET (Taraska et al., 2009)) was used to shed light on which
MODEL OF ATP HYDROLYSIS IN CLPX
Covalently connecting ClpX subunits to produce a
pseudohexamer was an important tool for studying how ATP
hydrolysis and activity is coupled in ClpX. Based on such
constructs and mutational studies, various working models such
A
C
B
D
FIGURE 2 I ATP hydrolysis mechanism and CoMET assay. (A) Early models of ATP hydrolysis mechanisms for ClpX. (B) nCoMET binding assay for detecting
nucleotide binding. When a nucleotide binds, quenching occurs. (C) cCoMET assay for detection of conformational changes. Quenching of dye indicates a subunit
is loadable, and is no quenching indicates the subunits is unloadable. (D) cCOMET (left) and nCoMET (right) results when low nucleotide binding affinity mutants
were introduced to subunits. (D) is adapted by permission from ELSEVIER Ltd: [Cell] (Stinson et al., 2013) copyright.
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Structural insights on ATP hydrolysis-driven mechanical work of AAA+ hexamers
states the hexamer’s subunits are in at various conditions (Figure
2B,C). It was found that when there are no nucleotides, ClpX
exists in a 4L:2U structure. But when sufficient nucleotides are
present, it changes to a 5L:1U structure. Such findings, which
were not evident in the crystal structures, implied that when
ClpX is working it changes its conformation very dynamically.
The same trend was observed even when ATP was replaced
with ADP or ATPγS, an ATP analog that is difficult is hydrolyze.
So, ClpX can bind nucleotides other than ATP, and thus release
of ADP binding of the proper nucleotide (ATP) are essential for
continuous functioning.
Then the team went on to produce a pseudohexamer by
dimerization of a chain of three ClpX subunits connected
covalently. Each ‘trimer’ construct consisted of two wildtype
subunits (W) and a low nucleotide affinity mutant (VI) (Martin
et al., 2008) in the middle. Applying the CoMET method again,
it was found that the subunits adjacent to the VI mutant have
different nucleotide binding affinities (Figure 2D left: cCoMET,
right: nCoMET). Specifically, it was found that the six subunits
follow the specific order empty-tight-weak-empty-tight-weak.
Furthermore, when two of the subunits were VI mutants or
additional ATP hydrolysis mutants (VIE) (Martin et al., 2008), the
hydrolysis rate and protein activity changed greatly depending
on ATP concentration. However, the trends were different from
that of the wild type pseudohexamer (Figure
3A). This indirectly showed that the subunits’
configurations in the hexamer are subject to
A
switching.
Based on such results, the ‘setting-resetting’
gradually translocated through 1 nm bursts (Figure 4B). This
implied that normally two subunits facing each other perform
work together, supporting the setting-resetting model.
When the extension traces of translocation were viewed in
detail, there were two alternating phases. There was a ‘dwell
phase’, where there was no extension for some time, and
a ‘burst phase’, where the substrate was translocated near
instantaneously. Notably, when ATP S was added, there was no
effect on the dwell phase (Figure 4B). Based on this observation,
it was proposed that ClpX follows the ‘power-stroke model’,
where phosphate ion (Pi) release and burst occurs concurrently
(Figure 4C).
However, this means that ADP release and ATP recharge both
occur during the dwell phase. To determine which of the steps is
>
rate-limiting, n = (< 𝜏𝜏 <> 𝜏𝜏−<
, a value that indicates the lower
𝜏𝜏 > )
min
2
2
2
bound for the number of rate-limiting steps was determined,
which turned out to be 2. Because addition of Pi analogs did not
affect nmin and the burst size, but the dwell phase increased
(Figure 4D), it was deduced that ADP release is the rate-limiting
step.
Almost all known homologs of ClpX have a well conserved
GYVG motif (Park et al., 2005), which directly binds with the
substrate during translocation (Figure 5A). Various mutations
were introduced to the covalently connected ClpX hexamer, to
model was proposed (Figure 3B) (Stinson et
al., 2013). In this model, ClpX is initially in
the 4L:2U state. Two ATP molecules bind to
the tight binding subunits, followed by two
more ATP binding to the weak binding sites,
resulting in the 5L:1U state. ATP hydrolysis
occurs at the tight binding sites, and a cycle
ends by the tight binding sites and weak
binding sites resetting.
TRANSLOCATION MECHANISM OF
CLPX USING SINGLE-MOLECULE
FORCE-SPECTROSCOPY
Many recent works utilize optical tweezers to
study the translocation process of ClpX (AubinTam et al., 2011; Rodriguez-Aliaga et al., 2016).
By observing the length change of a substrate
for ClpX, the degree and rate of translocation
can be monitored under various conditions
(Rodriguez-Aliaga et al., 2016) (Figure 4A).
In Rodriguez-Aliaga et al.’s work, substrates
translocated in 2 nm bursts when only ATP
was added. But when the concentration of
ATPγS was gradually increased, the substrate
4
B
FIGURE 3 I Effect of mutations and contemporary model of ClpX action. (A) ATP hydrolysis
and activity of low nucleotide binding affinity ClpX mutants. (B) The ‘setting-resetting’ model, the
most up-to-date model which takes account of all results so far. (A) is adapted by permission from
ELSEVIER Ltd: [Cell] (Stinson et al., 2013) copyright.
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Changwon Kim, Sang-Hyun Rah and Tae-Young Yoon
A
B
C
D
FIGURE 4 I Single molecule force spectroscopy of ClpX using optical tweezers. (A) Experiment schematic for optically tweezing the ClpX-ClpP complex
(left), and a typical translocation trace of a substrate (right). (B) Dwell duration and burst size distributions of translocation when ATPγS and ATP are both present.
(C) Expected scheme of ATP hydrolysis and recharge during translocation. The behavior seems to follow the p ower-stroke model. (D) Dwell duration and burst
sizes (left) and nmin (right) for various concentrations of Pi analog. Adapted by permission from Macmillan Publishers Ltd: [Nature Structural & Molecular Biology]
(Rodriguez-Aliaga et al., 2016) copyright.
control the size of the motif and the number of mutated subunits
(Figure 5B). When the interacting residue was smaller than the
wild type, the ATP hydrolysis rate increased and the translocation
velocity was faster, while for bulkier mutations the trend was the
opposite (Rodriguez-Aliaga et al., 2016). In all cases the burst
size was unaffected (Figure 5C). However, the translocation
to ATP hydrolysis ratio was most efficient for the wild type,
explaining how this sequence could be so well conversed during
evolution (Figure 5D).
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NSF`S FUNCTIONAL MECHANISM WITH SNAPS
AND SNARE
N-ethylmaleimide-sensitive factor (NSF) is a protein essential
for intracellular membrane trafficking (Wilson et al., 1989).
Membrane fusion is mediated by the assembly of soluble NSF
attachment protein receptor (SNARE) proteins into a single
SNARE complex (Fasshauer, 2003; Hayashi et al., 1994). The
role of NSF is disassembling the SNARE complex into individual
SNAREs for another membrane fusion event. Disassembly is
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Structural insights on ATP hydrolysis-driven mechanical work of AAA+ hexamers
A
C
B
D
FIGURE 5 I Mutation experiments on the GYVG motif of ClpX. (A) Location of the GYVG motif near the pore. (B) One to three mutations were introduced to
the motif to vary the ClpX pore size. (C) Burst size, dwell duration (left), ATPase rate, and velocity (right) for various pore sizes. (D) Of all the constructs studied, the
wildtype motif has the highest efficiency of converting ATP hydrolysis to translocation. Adapted by permission from Macmillan Publishers Ltd: [Nature Structural &
Molecular Biology] (ref. 16) copyright (2016).
assisted by soluble NSF attachment proteins (SNAP), which first
binds to the SNARE complex and recruits an NSF hexamer to the
SNAP-SNARE complex. When NSF binds to the SNAP-SNARE
complex, forming the so called ‘20S complex’, ATP hydrolysis is
initiated and the SNARE complex is disassembled (Figure 6E).
Although functionally distinct from ClpX, NSF is also formed
of three domains - an amino (N) domain that mediates binding
to substrates, and two AAA+ domains. Also, although there are
dozens of SNAREs in a eukaryote, there are very few isoforms
of NSF (Bock et al., 2001), implying various SNARE binding
capability. However, despite such similarities, the mechanism
behind NSF’s activity was found to be vastly different.
STRUCTURE OF 20S COMPLEX AND
INDIVIDUAL PROTEINS
There are at least 24 SNARE proteins in yeast and over 60 in
mammals, but we shall focus on neuronal SNAREs in this review.
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The neuronal SNARE complex is an archetypal four helix bundle
formed of target membrane SNAREs (t-SNAREs) synaptosomalassociated protein 25 kDa (SNAP-25) and syntaxin-1, and a
vesicle SNARE (v-SNARE) vesicle-associated membrane protein
2 (VAMP2). A SNARE complex typically has 16 hydrophobic
layers within the bundle, but at the central ‘zeroth’ layer, there
are ionic residues (one arginine and three glutamines) which are
conserved across the SNARE family (Sutton et al., 1998) (Figure
6A).
Of the three (α-,β-, γ -) SNAPs in cells, the one responsible
for neuronal SNARE disassembly is α-SNAP (Griff et al., 1992;
Whiteheart et al., 1993). α-SNAP is a protein formed of 14
α-helices and binds strongly to the SNARE complex using ionic
interactions (Figure 6B). The C-term of α-SNAP has negatively
charged residues which allow binding to one or two N domains of
NSF, which contains positive residues (Figure 6C). As discussed
above, an NSF monomer is composed of three domains. The N
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Changwon Kim, Sang-Hyun Rah and Tae-Young Yoon
A
B
C
D
E
FIGURE 6 I Structure of the 20S complex and its constituents. (A) Sequence of individual neuronal SNARE proteins and their layer positions. (A) is from [Ryu et
al., Science 2015, 347 (6229), 1485-1489.]. (B) Structure of α-SNAP, formed of 14 α-helices (PDB 1QQE). (C) α-SNAP can bind two NSF N domains via strong ionic
interactions. (D) The structure of an NSF hexamer and its three domains. (E) Structure of the 20S complex obtained by cryo-EM. Four a-SNAPs are attached to the
SNARE complex and NSF (PDB 3J96). (C) is adapted by permission from Macmillan Publishers Ltd: [Nature] (Zhao et al., 2015) copyright.
domain allows interaction with α-SNAP, and the D2 domain is
responsible for tight oligomerization of the monomers by binding
ATP. Finally, the D1 domain contains an AAA+ domain which
provides the energy for conformation change of the N domain via
ATP hydrolysis (Figure 6D).
Recently, Axel Brunger’s group solved the structure of the
NSF hexamer and the entire 20S complex by cryo-electron
microscopy (Figure 6I), providing critical insights to how the
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individual proteins are connected and may lead to SNARE
disassembly (Zhao et al., 2015). It was found that depending on
whether ATP or ADP is bound to the D1 domain, the N domain
is positioned either towards the SNARE complex or beside the
D2 domain, respectively. In addition, the overall form of the ATPbound NSF hexamer takes a right-handed ‘split-washer’ form,
while ADP-bound NSF takes an ‘open flat washer’ form (Figure
7A). Because the SNARE complex is bundled in a left-handed
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Structural insights on ATP hydrolysis-driven mechanical work of AAA+ hexamers
A
B
C
D
E
F
FIGURE 7 I Studies on the functional mechanism of NSF. (A) ATP bound NSF changes to the ADP bound state through a large conformation change of the N
domain, resulting in a configuration change of the D1 ring. (B) Structure of the D1 domain’s nucleotide binding pockets in the ATP and ADP state. If a nucleotide
is assumed to exist in the pocket in the ADP state, a clash occurs. (A) and (B) are adapted by permission from Macmillan Publishers Ltd: [Nature] (Zhao et al.,
2015) copyright. (C to F) SNARE disassembly reconstitution using total internal reflection microscopy. (C) Schematic for measuring SNARE disassembly by NSF.
(D) Fluorescently labeled SNAREs disappear only when α-SNAP, NSF, ATP and Mg2+ are all present. (E) Schematic for testing the role of hydrolysis of ATPs already
bound to NSF, i.e. one-round ATP hydrolysis. (F) One round-ATP hydrolysis is sufficient for complete SNARE complex disassembly. (C) to (E) are from [Ryu et al.,
Science 2015, 347 (6229), 1485-1489.].
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Changwon Kim, Sang-Hyun Rah and Tae-Young Yoon
orientation, the conformation change of NSF from the ATP state
to the ADP state is speculated to be well-suited for SNARE
complex disassembly. However, in the ADP state, the position
of the α7 helix in the D1 domain overlapped with the nucleotide
binding space. This implies that the ADP state structure
observed may be in fact be the Apo (empty) state (Figure 7B).
SINGLE-MOLECULE ASSAY WITH 20S
COMPLEX
Single molecule experiments on the 20S complex were mostly
done with total internal reflection fluorescence microscopy (Ryu
et al., 2015). First, SNARE complexes tagged with a fluorescence
dye,Areconstituted into liposomes, were immobilized to a quartz
A
surface. α-SNAP was injected, and unbound molecules were
washed out. NSF/ATP/Mg2+ (NAM) mixture injection followed
(Figure 7C). After washing out unbound molecules, the number
of fluorescent spots on the surface significantly decreased,
compared to when EDTA was included instead of Mg2+ (NAE) –
indicating that SNARE disassembly occurred (Figure 7E).
In a similar experiment, the 20S complex was formed by initially
adding NAE, followed by free molecule removal. Surprisingly,
when only Mg2+ was added to this state, disassembly occurred
(Figure 7E). Because the resulting disassembly level was
identical to the NAM condition, it could be inferred that SNARE
disassembly by NSF requires at most 6 ATP molecules, i.e. those
that were bound to the hexamer prior to 20S complex formation
A
B
B
B
C
C
ATP binding
C
APO state
ATP state
ATP binding
APO state
ADP release
(Spring-loaded)
ADP release
(Spring-loaded)
ATP state
ATP hydrolysis
ATP hydrolysis
ADP state
Spring-loaded
ADP state
ADP+Pi
Pi release
(Power-stroke)
ADP+Pi
Pi release
FIGURE 8 I Molecular model of NSF action. (A) Schematic for testing effect of free phosphate ions and Pi analogs. (B) Two possible molecular models for
(Power-stroke)
Spring-loaded
conversion of ATP hydrolysis to disassembly by NSF from [Ryu et al., Science 2015, 347 (6229), 1485-1489.]. (C) The ATP hydrolysis cycle and steps which are
coupled to mechanical work.
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Structural insights on ATP hydrolysis-driven mechanical work of AAA+ hexamers
(Figure 7F).
As discussed, such high energetic efficiency is likely possible
due to the SNARE complex’s left handed helical structure
and the right-handed conformational change of NSF’s N
domain. Analogous to the experiments done for ClpX, when Pi
analogs were added to the surface immobilized 20S complex
disassembly assay, the dwell time between Mg2+ injection and
SNARE disassembly significantly increased (Figure 8A). Because
Pi analogs stabilize the hydrolysis intermediate state, such
results suggested that the dwell time is a result of NSF being
unable to change its conformation from the ATP state to the ADP
(or Apo) state.
This behavior was further clarified by the use of magnetic
tweezers. In this assay, the soluble fraction of the SNARE
complex was weakly pulled at the C-termini by tethering to
the surface and a magnetic bead. Although the force applied
was weak enough to produce no perturbation to the SNARE
complex at all, addition of α-SNAP and NAM led to an extremely
fast (<20 ms) burst of complete disassembly, instead of a
processive disassembly process (Min et al., 2013) (figure not
shown). Summing up, NSF initially stores all the energy from
ATP hydrolyses in its ATP conformation, and releases it nearly
instantaneously as it changes to the ADP conformation and
disassembles the SNARE complex – which supports the ‘springloaded’ model.
TWO MOLECULAR MODELS OF AAA+ ATPASE
OPERATION
ClpX and NSF are examples of the two molecular models of how
AAA+ ATPases link ATP hydrolysis to their function, the powerstroke model and the spring-loaded model (Figure 8B). Generally,
when an ATP binds to an empty ATPase, it is converted to the
ATP state. This is followed by ATP hydrolysis, resulting in ADP
and a Pi bound to the ATPase. Because the free energy change
of this cleavage process is too small, this step is not associated
with mechanical work. So, the two models differ in behavior that
follows ATP cleavage. In the power-stroke model, mechanical
work of a subunit is directly coupled to a Pi release event. On
the other hand, in the spring-loaded model, the Pis are released
from the subunits without producing work. The ATPase cannot
convert to the ADP state as the substrate resists being unfolded.
When all the Pis are released and a thermal fluctuation allows
crossing of the energy barrier, conformation change is triggered,
which produces mechanical work. Whichever model is followed
by an ATPase, ADP is ultimately released from the ATPase and
the cycle repeats (Figure 8C).
CONCLUSION AND FUTURE CHALLENGES
Exciting technological developments have shed light on the
mechanism of AAA+ proteins in unprecedented detail - yet
there are many interesting questions remaining to be answered.
For NSF, it is known that conformation change is difficult when
nucleotides are bound. Also, due to the fact that the size is about
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twice as big as ClpX, it is difficult to link all six subunits using
peptide linkers. Hence it is difficult to introduce well-defined
point mutations to each subunit, and determine whether less
than six ATP molecules are sufficient for SNARE disassembly.
For ClpX, the setting-resetting model is gaining support, but it is
uncertain whether the resetting step is sequential or probabilistic.
Also, further study is required to elucidate how ATP hydrolyses
occur in symmetrical fashion. Lastly, it would be interesting to
understand how different proteins from the same AAA+ family
can function by vastly different molecular models of action.
ACKNOWLEDGEMENTS
This work was supported by the National Creative Research Initiative
Program funded by the NRF (Center for Single-Molecule Systems Biology
NRF-2011-0018352 to T.-Y.Y.). C.K. and S.-H.R. were supported by
Institute for Basic Science (IBS; IBS-R0216-D1).
AUTHOR INFORMATION
The authors declare no potential conflicts of interest.
Original Submission: Dec 14, 2016
Revised Version Received: Jan 31, 2017
Accepted: Feb 7, 2017
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H.W., and Sui, S.F. (2012). Structural characterization of full-length NSF
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