Structure

ArticleHigh-Speed Atomic Force Microscopic Observationof ATP-Dependent Rotation of the AAA+ Chaperone p97
Kentaro Noi,1,2,7 Daisuke Yamamoto,3,6,8 Shingo Nishikori,1,6,9 Ken-ichi Arita-Morioka,1,2 Takayuki Kato,5Toshio Ando,3,4,6 and Teru Ogura1,2,6,*
1Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics
2The Global COE (Cell Fate Regulation Research and Education Unit)
Kumamoto University, Kumamoto 860-0811, Japan
3Department of Physics
4Bio-AFM Frontier Research Center
Kanazawa University, Kanazawa 920-1192, Japan
5Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan
6Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo 102-0075, Japan
7Present address: Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University,
Osaka 560-8531, Japan
8Present address: Department of Applied Physics, Fukuoka University, Fukuoka 814-0180, Japan
9Present address: Otsuka Pharmaceutical Co., Ltd., Tokushima 771-0192, Japan*Correspondence: ogura@gpo.kumamoto-u.ac.jp
http://dx.doi.org/10.1016/j.str.2013.08.017

SUMMARY

p97 (also called VCP and CDC-48) is an AAA+ chaperone, which consists of a substrate/cofactorbinding N domain and two ATPase domains (D1and D2), and forms a homo-hexameric ring. p97
plays crucial roles in a variety of cellular processes
such as the ubiquitin-proteasome pathway, theendoplasmic reticulum-associated protein degradation, autophagy, and modulation of protein aggregates. Mutations in human p97 homolog VCP arelinked to neurodegenerative diseases. The key
mechanism of p97 in these various functions has
been proposed to be the disassembly of protein
complexes. To understand the molecular mechanism of p97, we studied the conformational changesof hexameric CDC-48.1, a Caenorhabditis elegans
p97 homolog, using high-speed atomic force microscopy. In the presence of ATP, the N-D1 ring repeatedly rotates 23 ± 8 clockwise and resets relative to
the D2 ring. Mutational analysis reveals that this rotation is induced by ATP binding to the D2 domain.

INTRODUCTION
AAA+ (ATPases associated with diverse cellular activities) family
proteins are ATP-dependent molecular chaperones, which exert
mechanical work on macromolecular substrates, such as unfolding of proteins and disassembly of multisubunit protein complexes (Ogura and Wilkinson, 2001; Hanson and Whiteheart,2005). It has been proposed that AAA+ chaperones undergo
conformational changes during the cycle of ATP hydrolysis,
and that the conformational changes provide their mechanicalwork on substrates. AAA+ proteins contain one (type I) or two(type II) conserved AAA+ domains, consisting of the ATPasedomain with the Walker A (ATP binding) and Walker B (ATPhydrolysis) motifs followed by an a-helical C-domain (Ammelburg et al., 2006). AAA+ domains form a ring-shaped hexamer
and thus there are 6 and 12 ATPase active sites in the type I
and type II AAA+ proteins, respectively.
p97 (also known as VCP in mammals, CDC-48.1 and CDC48.2 in Caenorhabditis elegans, and Cdc48p in yeast) is a highly
conserved and abundant type II AAA+ protein. p97 plays essential roles in a number of cellular processes including membrane
dynamics, cell cycle control, the ubiquitin-proteasome system,
autophagy, and modulation of protein aggregates (for recent reviews, Stolz et al., 2011; Yamanaka et al., 2012; Meyer et al.,2012; Buchberger, 2013). Human p97 (VCP) has been linked toneurodegenerative disorders, hereditary inclusion body myopathy associated with Paget disease of bone and frontotemporaldementia (Watts et al., 2004; Weihl et al., 2009), and amyotrophic
lateral sclerosis (Johnson et al., 2010). TAR DNA-binding protein
43-positive inclusions are accumulated in neuronal cells of thepatients with both diseases (Johnson et al., 2010).
p97 consists of an N-terminal substrate/adaptor-binding
domain followed by two AAA+ domains (D1 and D2) and forms
a homo-hexameric double-ring structure. It has been shownthat D2 mediates major ATPase activity (Song et al., 2003; DeLaBarre et al., 2006), and that the ATPase activity of D2 is critical forbiological functions (Song et al., 2003; DeLaBarre et al., 2006;
Esaki and Ogura, 2010; Nishikori et al., 2011). A quantitative
analysis of nucleotide-binding to both D1 and D2 domains of
rat p97 indicates that ATPgS, an ATP analog, binds to D1 andD2 with similar affinity, but ADP binds to D1 with higher affinity
than to D2, which is consistent with the higher ATPase activity
in D2 (Briggs et al., 2008). Stoichiometric measurements suggestthat both ADP and ATPgS can saturate all six nucleotide bindingsites in D1, but that only three to four of the six D2 sites can bind
ATPgS simultaneously, and that ATPgS binding to the D2
domain triggers a downstream cooperative conformationalchange of at least three monomers (Briggs et al., 2008). The

1992 Structure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reservedStructure
Rotation of p97 Analyzed by High-Speed AFM

Figure 1. AFM Image of C. elegans CDC-48.1 Hexamers and Its
Comparison with an Image of Mammalian p97 Simulated from the
Crystal Structure(A) A wide-area AFM image of CDC-48.1 hexamers without nucleotide. Scalebar, 50 nm.
(B) A filtered AFM image of a CDC-48.1 hexamer. Scale bar, 5 nm.
(C) The N-terminal side of the crystal structure of mouse apo p97 (PDB ID
3CF3; Davies et al., 2008). The N and D1 domains are magenta and blue,
respectively. Scale bar, 5 nm.
(D) A simulated AFM image of the p97 hexamer constructed from the structure
in C. Scale bar, 5 nm.
(E) A wide-area AFM image of DN CDC-48.1 hexamers without nucleotide.
Scale bar, 50 nm. The brightness of all AFM images in this figure represents the
sample height but is not linearly set to highlight the top surface structure.
See also Figure S1.

D2 ATPase activity shows positive cooperativity (Nishikori et al.,2011). We recently found, using the yeast Saccharomyces cerevisiae, that cells with Cdc48p mutants deficient in ATP binding of
D1 are able to grow, suggesting that ATPase deficiency of D1
does not affect the vital D2 ATPase activity in terms of essentiality (Esaki and Ogura, 2010). A systematic analysis of recombinant CDC-48.1 of C. elegans was performed to characterize
the ATPase activity of D1 and D2 of CDC-48.1. Like mammalianp97, D2, but not D1, of CDC-48.1 showed strong ATPase activity
with positive cooperativity at physiological temperature. The
ATPase activity itself and its cooperativity of D2 are differently
affected by ATPase-deficient mutations in D1. Interestingly,
magnitude of the cooperativity of D2 is well related to growthrate of yeast cells. The D2 ATPase activity of p97 is operated
in a nonstochastic fashion, and its positive cooperativity is critical for viability of the cell (Nishikori et al., 2011).

Crystal structures of p97 in different nucleotide states havebeen solved (DeLaBarre and Brunger, 2003; DeLaBarre et al.,2006; Huyton et al., 2003). Cryo-electron microscopy (EM) andsmall-angle X-ray scattering (SAXS) analyses also revealed conformations of p97 with different nucleotides (Zhang et al., 2000;
Rouiller et al., 2000, 2002; Beuron et al., 2003; Davies et al.,2005). From these data, several models for conformationalchanges of p97 during the cycle of ATP hydrolysis have been
proposed (for a review, see Pye et al., 2006). However, there
have been no studies providing information about the dynamics
of native p97 functioning in solution.
To depict conformational changes of p97 during the cycle of
ATP hydrolysis in solution, we applied high-speed atomic force
microscopy (AFM), a newly developed video-rate imaging technique, which enables to capture high-resolution images in millisecond time scales. By using high-speed AFM, several protein
molecules such as myosin V, F1-ATPase, and cellulase wereanalyzed and their linear or rotary movements were revealed inrecent studies (Kodera et al., 2010; Uchihashi et al., 2011; Igarashi et al., 2011).
In this paper, we observed a C. elegans p97 CDC-48.1 immobilized on a mica surface with high-speed AFM. Clear images of
CDC-48.1 ring-shaped hexamers with a narrow central pore
were depicted and, in the presence of ATP, rotational movements of the D1 ring relative to the D2 ring were observed. The
D1 ring repeatedly rotated 23 ± 8 clockwise and returned tothe original position. This rotational movement was strictly
dependent on ATP, and ADP did not induce the rotation of
wild-type CDC-48.1. Mutational analysis revealed that the ringrotation was induced by ATP-binding to the D2 domain. The
molecular mechanism of p97, in which ATP-binding to the D2
domain induces conformational change, that in turn gives a twistat the linker between the N-D1 and D2 domains, has beensuggested. Taken together, our demonstration of functioning
p97 with high-speed AFM provides insight into the molecularmechanism of p97.
RESULTS
High-Speed AFM Observation of a C. elegans p97
ATPase, CDC-48.1
Purified N-terminally His-tagged wild-type CDC-48.1, a
C. elegans p97 ATPase, was directly immobilized on a surfaceof mica (Figure S1A available online), and was observed withhigh-speed AFM. Images of observed p97 particles were apparently homogeneous hexameric rings of 18.3 ± 1.7 nm in diameter (Figures 1A and 1B). The observed image of the CDC-48.1
hexamer (Figure 1B) looks similar to an image of the N-D1 sideof mouse p97 (Figure 1D), which was simulated from the crystal
structure (Figure 1C; Davies et al., 2008), and its size is roughly
consistent with that of the simulated image. To confirm thenotion that the N-D1 side is observed under the conditions
used here, a mutant CDC-48.1 lacking the N domain was also
purified and observed with high-speed AFM. We found thatimages showed homogeneous hexameric rings of 15.3 ±1.6 nm in diameter (Figure 1E), which is significantly smaller
than that of the full-length wild-type. Considering these lines ofevidence together, we conclude that the C-terminal part of
CDC-48.1 was, at least predominantly, immobilized, andStructure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reserved 1993

Structure
Rotation of p97 Analyzed by High-Speed AFM

Figure 2. ATP-Dependent Rotational Movements of Wild-Type CDC-48.1 Hexamers

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therefore that the N-D1 side was depicted when CDC-48.1 was
immobilized directly. To further confirm this notion, we tried toimmobilize CDC-48.1 by using its N-terminal His-tag. The
N-terminally His-tagged CDC-48.1 was immobilized on a surfaceof streptavidin crystals through binding of His-tag to Ni-NTAbiotin attached to streptavidin (Figure S1C). Images showedrings of 15.0 ± 1.4 nm in diameter, which is smaller than thatof the directly immobilized full-length wild-type, and fits withthe size of the D2 ring reasonably well.
As shown in Figure 1, the images of the N-D1 side of CDC-48.1
hexamers immobilized directly on a surface of mica clearly indicate a 6-fold symmetric ring with a narrow central pore. The
height of CDC-48.1 hexamers was 5.7 ± 0.7 nm, which wassignificantly shorter than those (8.4–11.0 nm) of the crystal structures of mammalian p97. Therefore, human p97 was purified andobserved. The height of the human p97 ring was 6.4 ± 0.7 nm,
which was significantly taller than that of the CDC-48.1 ring, but
still shorter than those of the crystal structures of rat p97. The
reason for the difference in height between AFM images andcrystal structures is uncertain. On the other hand, the imagesof the D2 side of CDC-48.1 hexamers were enough for measurement of diameter as described above, but unfortunately not
sufficient for precise analysis of shape, perhaps due to lateral
diffusion of the lipid bilayer. Therefore, we decided to analyze(A) Successive AFM images of a wild-type CDC48.1 hexamer taken in the presence of 3 mM ATP(Movie S1). Three diametric lines colored differently are indicated in the first image (0.0 s). Only
one of these lines (black) is indicated in other images for simplicity.
See also Figure S2A.
(B–E) Time course of angular variation of diametriclines in the successive images shown in (A) is
shown. Clockwise rotation is indicated by plus
degrees, and counterclockwise one is indicatedby minus degrees. Graphs of three lines areoverlaid in (B), and each is separately shown in (C),(D), and (E).
(F and G) Two representative 10 s time courses
show repeated rotational movements. The first
2.5 s graph of (F) corresponds to that of (C;note the difference in time scale). Longer timecourses containing these two examples andadditional time courses are shown in Figures
S2B–S2E.
(H) Histogram of the angle of diametric lines ofwild-type CDC-48.1 hexamers in the presence of
3 mM ATP. Number of hexamers observed = 9;
total observation period = 107.8 s. Red line,
double-Gaussian fit result; blue lines, Gaussiancomponents in double-Gaussian fit. Peak value
represents mean ± SD analyzed and calculatedusing Origin 6.0 software.
See also Figures S2F–S2I and Movie S2 formovements of wild-type CDC48.1 hexamers
lacking one protomer.

conformational changes of the N-D1 side of CDC-48.1 hexamers
in detail.
ATP Induces Rotation of the N-D1 Ring of CDC-48.1
To achieve high-resolution video-rate observation of CDC-48.1
hexamers, the images of CDC-48.1 were taken with a frame
capture time of 100 ms unless otherwise specified. In the presence of 3 mM ATP, rotational movements, which were distinguishable from irregular Brownian motions, were repeatedly
observed (Figure 2; Movie S1) for roughly 30%–40% of hexameric rings of CDC-48.1. In a series of typical images (Figure 2A),
changes in the angle of three diametric lines that go throughthe centers of opposite D1 subunits were measured (Figures
2B–2E). The N-D1 ring rotated 23 ± 8 clockwise relative to
the D2 ring immobilized to a mica surface, stayed in the rotatedposition for 0.1 s or more, and returned to the original position(Figures 2F and 2G). In the rotated state, all three diametric lines
are similarly at the rotated position (Figures 2C–2E), indicatingthat the 6-fold symmetry of the N-D1 ring is essentially unchanged during the course of movements. Reliability of theabove analysis has been verified by the image analysis using
EMAN1’s boxer and SPIDER programs, both of which arefrequently applied for analysis of electron microscopic images(Figure S2A).

1994 Structure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reservedStructure
Rotation of p97 Analyzed by High-Speed AFM

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Figure 3. Movements of Wild-Type CDC48.1 Hexamers in the Absence of Nucleotide
and in the Presence of 3 mM ADP

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Time (s)Distributions of the line angle measured for nine independent
hexamers, which showed significant rotational movements during the observation (total observation period was 107.8 s; Figures S2B–S2E for four out of nine), are shown in Figure 2H. There
are two peaks at positions 0 (resting state) and 23 (rotatedposition). From the distributions, the rotational movement seems
to be a single 23 step with no evidence of substeps. Since theperiodicity in a hexamer is 60 , a 23 clockwise rotation appears
equivalent to a 37 counterclockwise rotation. To discriminate
between these possibilities, it is necessary to label a hexamer
asymmetrically. During the observation with high-speed AFM,
we noticed that some hexamers accidentally lost one subunit
and continued to move for a couple of seconds, and that they
finally disassembled and disappeared from the visual field (Figures S2F–S2I; Movie S2). From these results, we conclude thatthe rotation is 23 ± 8 in the clockwise direction.
Under the present conditions, we are unable to observe
the conformational changes of the D2 domain. As describedlater, the observed rotational movement depends on ATPbinding to the D2 domain. Therefore, it is most likely that ATPbinding to the D2 domain induces its conformational changes,
leading to the clockwise rotation of the D2 ring relative to the
N-D1 ring when observed from the bottom. This movement results in the clockwise rotation of the N-D1 ring relative to theD2 ring immobilized on a surface of mica when observed fromthe top.
Rotation of the N-D1 Ring Depends on ATP
To know the nucleotide dependency of the rotational movementof the N-D1 ring, we observed CDC-48.1 in the absence of nucleotide and the presence of 3 mM ADP. Because the symmetry of
the hexamers was again unchanged, changes in the angle of only
one diametric line were shown for simplicity. In both cases, large
rotational movements, as in the case of the presence of ATP,were not observed (see Figures 3A–3C for no nucleotide and Figures 3D–3F for ADP; Figure S3; Movies S3 and S4). Therefore,

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AFM images are taken and analyzed as in Figure 2.
(A and B) Two representative 10 s time courses ofwild-type CDC-48.1 hexamers in the absence of
nucleotide are shown (Movie S3).
(C) Histogram of the angle of diametric lines ofwild-type CDC-48.1 hexamers in the absence of
nucleotide. Number of hexamers observed = 10;
total observation period = 164.8 s. Red line, singleGaussian fit result. Longer time courses containing
these two examples and additional other timecourses are shown in Figures S3A–S3D.
(D and E) Two representative 10 s time courses ofwild-type CDC-48.1 hexamers in the presence of
3 mM ADP are shown (Movie S4).
(F) Histogram of the angle of diametric lines ofwild-type CDC-48.1 hexamers in the presence of
ADP. Number of hexamers observed = 10; total
observation period = 139.5 s. Red line, singleGaussian fit result. Longer time courses containing
these two examples and additional time courses
are shown in Figures S3E–S3H.

we conclude that the 23 ± 8 clockwise rotation of the N-D1
ring strictly depends on the presence of ATP.
Lower ATP Concentration Decreases Abundance ofRotated StateAFM experiments were performed in the presence of a lower
concentration of ATP, 0.3 mM (Figure 4). The clockwise rotations
were again observed (Figures 4A and 4B; Figure S4), and thepeak of the rotated position was 19 ± 10 (Figure 4C). The
peak of the rotated position (the number of frames in the rotatedstate) was significantly lower than that in the presence of 3 mM
ATP (Figure 2H). To compare the kinetics at different concentrations of ATP precisely, we measured dwell time in the restingstate and lifetime of the rotated state (Figures S4E–S4H). Thesetime distributions are fitted to sums of two exponential decayfunctions. While dwell time in the resting state, which corresponds to the time waiting for ATP binding, in the presence of
0.3 mM ATP was slightly longer than that in the presence of
3 mM ATP (Figures S4E and S4F), lifetime of the rotated state,
which corresponds to the time ATP remains bound, was significantly shortened by the decrease of the ATP concentration as
evident by an 80% decrease of preexponential factor A2 oftime constant t2 (Figures S4G and S4H).
Rotation of N-D1 Depends on Binding of ATP to the D2DomainWhich domain is responsible for the rotational movement?
Does ATP binding induce it, or is ATP hydrolysis required? To
address these important questions, we analyzed two mutant
CDC-48.1 proteins, CDC-48.1K530A and CDC-48.1E584Q, withhigh-speed AFM. It has been indicated that the D2 domain of
p97 shows major ATPase activity and the D1 domain, which is
responsible for stable hexamerization, shows only weak
ATPase activity (Song et al., 2003; DeLaBarre et al., 2006). Crystal structural analysis has indicated that purified p97 hexamers
contain six ADP bound to the D1 domain (DeLaBarre andStructure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reserved 1995

Structure
Rotation of p97 Analyzed by High-Speed AFM

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Figure 4. Movements of Wild-Type CDC48.1 Hexamers in the Presence of 0.3 mM
ATPC

AFM images are taken and analyzed as in Figure 2.
(A and B) Two representative 10 s time courses ofwild-type CDC-48.1 hexamers in the presence of
0.3 mM ATP are shown.
(C) Histogram of the angle of diametric linesof wild-type CDC-48.1 hexamers in thepresence of 0.3 mM ATP. Number ofhexamers observed = 10; total observation
period = 179.8 s. Red line, double-Gaussian
fit result; blue lines, Gaussian components indouble-Gaussian fit. Peak value represents mean ± SD. Longer time courses containing these two examples and additional time courses are shown in
Figures S4A–S4D.
See also Figures S4E–S4H for histograms of dwell time in the resting state and lifetime of the rotated state.

Brunger, 2003; Huyton et al., 2003). It has also been shown that
ATP binding to the D2 domain induces conformational changesof p97 (Zhang et al., 2000; Rouiller et al., 2000). Therefore, it isreasonable to assume that the rotational movement is inducedby the ATP binding to the D2 domain. CDC-48.1K530A and
CDC-48.1E584Q are Walker A and Walker B mutants of the D2
domain, which are defective in ATP-binding and ATP-hydrolysis,
respectively.
As expected, Walker A mutant CDC-48.1K530A did not show
the rotational movement in the presence of ATP (Figures 5A–
5C for ATP, and Figures 5D–5F for ADP; Figure S5), indicatingthat the ATP binding to the D2 domain is essential for the rotational movement. To know whether ATP hydrolysis is required
for the rotational movement, we then observed Walker B mutant
CDC-48.1E584Q in the presence of 3 mM ATP by high-speed
AFM. Images showed repeated rotational movements of the
N-D1 ring of CDC-48.1E584Q (Figures 6A–6C; Figures S6A–
S6D), indicating clearly that ATP binding, but not hydrolysis, isnecessary to induce the rotational movement. We then also
observed this mutant in the presence of 3 mM ADP. Surprisingly,
the N-D1 domain of CDC-48.1E584Q rotated clockwise repeatedly (Figures 6D–6F; Figures S6E–S6H). The observed rotation
angles of the mutant CDC-48.1E584Q in the presence of ATP
and ADP were 20 ± 6 (Figure 6C) and 22 ± 4 (Figure 6F),
respectively, and was similar to that of wild-type CDC-48.1 inthe presence of ATP (23 ± 8 ).
DISCUSSION
We have successfully observed and captured video-rate imagesof nucleotide-induced conformational changes of a multifunctional molecular chaperone CDC-48.1, a C. elegans p97
homolog, in solution by high-speed AFM. The observed conformational changes were mainly forward clockwise and backward counterclockwise rotational movements between the
N-D1 and D2 hexameric rings. Mutational analysis indicatesthat ATP-binding to the D2 domain induces the clockwise
23 ± 8 rotation of the N-D1 ring relative to the D2 ring; in otherwords, the counterclockwise rotation of the D2 ring relative to the
N-D1 domain when observed from N-D1. Because ATP binding
to the D2 domain is responsible for this rotation, it is reasonable
to assume that the observed conformational changes are
induced by changes in the D2 domain. Unfortunately, however,in the present study conformational changes in the D2 ringwere not seen, because the D2 domain was immobilized onto
a surface of mica. Rotational movements between the N-D1
and D2 rings of p97 have been suggested by cryo-EM and
SAXS studies (Zhang et al., 2000; Davies et al., 2005). Our
high-speed AFM data are essentially consistent with SAXS
data by Davies et al. (2005). It should be emphasized that ourstudy reveals ATP-dependent dynamic conformational changesof active p97 in an aqueous solution in real time.
It has been suggested that p97 promotes segregation of substrate proteins and disassembly of protein complexes. Repeatedback and forward rotational movements of p97 may account for,at least in part, mechanical force in such processes. In contrast
to AAA+ regulatory subunits of ATP-dependent proteases suchas ClpA and ClpX, which are often referred to as unfoldases,
there has been no convincing evidence for a threading mechanism, in which substrates are completely unfolded by threading
through the central pore, for p97, although it has recently beensuggested that archaeal Cdc48 homologs thread substrates as
discussed below. Instead, at least, two distinct mechanisms
have been proposed for p97; a ‘‘D2 in – D2 out’’ model, in which
substrates enter and exit the pore at the D2 domain, and a ‘‘surface only’’ model, in which the pronounced domain movementsof p97 during the cycle of ATP hydrolysis segregate or extract
substrates (Buchberger, 2013). Our present study clearly demonstrates that p97 undergoes obvious domain movements,and supports the surface-only model for the p97 activity. Importantly, however, our present data do not exclude any othermodels. In fact, recent studies suggest that archaeal Cdc48
homologs can thread substrates (Barthelme and Sauer, 2012,2013; Forouzan et al., 2012).
From crystal structural and cryo-EM studies, it has also beensuggested that the N domain of p97 is highly mobile (Rouiller
et al., 2002; DeLaBarre and Brunger, 2003). We have carefully
analyzed changes in the shape and height of the N-D1 ring during the rotational movements in the presence of ATP, but wehave failed to detect any significant differences in them, indicating that motions of the N domain are minimum or so rapid
and below the limit of detection, if any.
There are six identical ATPase domains in the D2 hexamericring. Briggs and colleagues have demonstrated that only three
to four of the six D2 sites can bind ATPgS simultaneously, andthat ATPgS binding triggers a cooperative conformationalchange of at least three D2 monomers (Briggs et al., 2008).
They also found that ATPgS binding to D2 is consistent with a1996 Structure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reservedStructure
Rotation of p97 Analyzed by High-Speed AFM

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Figure 5. Movements of Walker A Mutant
CDC-48.1K530A Hexamers in the Presenceof 3 mM ATP or ADP

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Time (s)two-site model with high- and low-affinity sites. For simplicity,
we assume here that four ATP molecules bind to the D2 ring
maximally and that two ATP molecules bind to D2 with a highaffinity, then an additional two ATP molecules bind with a low
affinity (Figure 7 for models). Our video imaging of CDC-48.1 inthe presence of ATP shows that the rotation of the N-D1 ring relative to the D2 ring is a single step event, indicating that this rotation seems to occur suddenly and to a fixed extent (23 ± 8 )upon binding of ATP to the D2 domain. We also predict thatthe binding of the third ATP induces the 23 ± 8 clockwise rotation of the D2 ring, and that the D2 ring with the bound three tofour ATP molecules stays in the rotated position. This prediction
is consistent with conformational changes determined by SAXS
analysis by Davies and colleagues (Davies et al., 2005). Theyobserved the 20 clockwise rotation of the D2 ring upon saturation by AMP-PNP.
ATP hydrolysis may occur in a coordinated manner or stochastically. We have recently reported that the ATPase activityof D2 is cooperative, and that this cooperativity is critical foressential functions of p97 (Nishikori et al., 2011). Model WT (Figure 7) implies that two or four ATP molecules are hydrolyzed
simultaneously, and it suggests a cooperative mode of ATPhydrolysis. This model also suggests that the D2 ring returns to
the original position (resting state) upon the hydrolysis of ATP,since the number of ATP molecules decreases to two or zero.
The histogram of lifetime of wild-type in the rotated state in thepresence of 3 mM ATP fits to a sum of two exponential curves
with preexponential factors of A1 = 353 s1 and A2 = 59 s1
and time constants of t1 = 0.066 s and t2 = 0.32 s (Figure S4G;
Table S1). Two exponential distributions were statistically significant judging from the F-test result (p < 0.01). According to Model
WT, rate constants can be determined as described in the Experimental Procedures.
Rate constants obtained for wild-type in the presence of 3 mM
ATP were k-2 = 10.0 s1, k3 = 4.7 s1, and k-3 = 2.1 s1 (Figure 7).

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AFM images are taken and analyzed as in Figure 2.
(A and B) Two representative 10 s time courses of
CDC-48.1K530A hexamers in the presence of 3 mM
ATP are shown.
(C) Histogram of the angle of diametric lines of
CDC-48.1K530A hexamers in the presence of 3 mM
ATP. Number of hexamers observed = 10; total
observation period = 82.6 s. Red line, singleGaussian fit result. Longer time courses containing
these two examples and additional time courses
are shown in Figures S5A–S5D.
(D and E) Two representative 10 s time courses of
CDC-48.1K530A hexamers in the presence of 3 mM
ADP are shown.
(F) Histogram of the angle of diametric lines of
CDC-48.1K530A hexamers in the presence of 3 mM
ADP. Number of hexamers observed = 10; total
observation period = 97.9 s. Red line, singleGaussian fit result. Longer time courses containing
these two examples and additional time courses
are shown in Figures S5E–S5H.

Preexponential factors and time constants calculated using
these values in the presence of 3 mM or 0.3 mM ATP wereconsistent with the experimentally determined values (Table
S1). Therefore, Model WT accounts for changes in the distribution of lifetime of the rotated state.
Do the observed repeated clockwise and counterclockwise
rotational movements represent the movements during cycles
of ATP hydrolysis? Because a Walker B motif mutant
CDC-48.1E584Q, which is defective in ATP hydrolysis in the D2
domain, also shows frequent rotational movements as similar
as wild-type CDC-48.1 (Figure 6), it is evident that ATP binding
and dissociation significantly contribute to repeated rotational
movements of wild-type hexamers. In Model WT, hexamers instate 2 are converted to those in the resting state either by dissociating one ATP molecule indicated as state 1 or by hydrolyzing
ATP molecules indicated as state 4. Using the rate constantsk2, k3, k3, and k4 of wild-type in the presence of 3 mM ATP,ratio of hexamers that underwent ATP hydrolysis in the D2
domain was calculated to be 19%, suggesting that the majorityof observed movements of wild-type hexamers are induced by
ATP binding and dissociation under the conditions used. Immobilization of CDC-48.1 to a surface of mica may affect its ATPase
activity. Indeed, it was found that CDC-48.1 immobilized to mica
showed a reduced ATPase activity, approximately one fifth ofthat measured in solution (data not shown). Frequent dissociation of the bound ATP molecules simply induces repeatedrotational movements of hexamers even in the case of
CDC-48.1E584Q. Under physiological conditions, CDC-48.1E584Q
would remain rotated longer, because it is unable to hydrolyze
ATP and dissociation of ATP would be much slower. Wild-type
CDC-48.1 can hydrolyze ATP, and therefore it would return tothe resting state predominantly by hydrolysis of ATP. The cycleof ATP hydrolysis ensures frequent repeated back and forthrotational movements, which may produce mechanical force todisassembly of substrate protein complexes.

Structure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reserved 1997

Structure
Rotation of p97 Analyzed by High-Speed AFM

60

40

40

20

20

120

Number of frames

60

0

0

-20

-20

-40

-40

-60

-60
0

2

4

6

8

80

2

4

6

8

20 ± 6°

40

0
0

10

10

-40

40

40

20

20

Angle (°)

60

0

20

40

F

E
60

-20

0

400

Number of framesD-20

Angle (°)

Time (s)Time (s)Angle (°)

Figure 6. Walker B Mutant CDC-48.1E584Q
Hexamers Rotate in the Presence of 3 mM
ATP or even ADP

C

B

Angle (°)

Angle (°)

A

300

0

200

-20

100

-40

-40

-60

-60
0

2

4

6

Time (s)

8

10

0

2

4

6

8

10

22 ± 4°0-40Time (s)In this respect, recent findings on N-ethylmaleimide-sensitive
factor (NSF), another type II AAA+ chaperone, which disassembles the soluble NSF attachment protein (SNAP) receptor(SNARE) complex in an ATP-dependent manner, are highly
intriguing. Using single-particle cryo-EM and negative-stain
EM, Chang and colleagues (Chang et al., 2012) found that ATPhydrolysis induces an 22 counterclockwise rotation of theD1 ring relative to the D2 ring, and they suggest that this rotationis related to a possible mechanism for converting chemicalenergy from ATP hydrolysis into mechanical work in the SNARE
complex disassembly (Chang et al., 2012). Because the D1 ringof p97 rotates 23 ± 8 clockwise upon ATP-binding and returns
to the original position (a 23 ± 8 counterclockwise rotation)
upon ATP hydrolysis as described above, both the directionand extent of the rotation in NSF upon ATP hydrolysis are fully
consistent with those in p97, suggesting that the molecularmechanisms underlying disassembly of protein complexes by
NSF and p97 may be fundamentally similar. It should be noted,
however, that the catalytically active ATPase domains aredifferent in NSF and p97. D1 is active and D2 is inactive in
ATP hydrolysis in NSF, while D2 is efficient and D1 is inefficientin p97.
Rate constants obtained for CDC-48.1E584Q in the presence of
3 mM ATP were k2 = 8.2 s1, k3 = 0.43 s1, and k3 = 0.72 s1(Figure 7; Table S1; Model E584Q). Rate constants k3 (0.43 s1)
and k-3 (0.72 s1) are roughly one tenth and one third of the wildtype values (4.7 s1 and 2.1 s1, respectively), whereas rate
constant k2 (8.2 s1) is similar to the wild-type value(10.0 s1). A simple explanation for CDC-48.1E584Q is that thebinding affinity of the fourth ATP to the mutant is much lower
than that of wild-type. Glu584 is the catalytic glutamate in D2

-20

0

20

40

Angle (°)

AFM images are taken and analyzed as in Figure 2.
(A and B) Two representative 10 s time coursesof CDC-48.1E584Q hexamers in the presence of
3 mM ATP.
(C) Histogram of the angle of diametric lines of
CDC-48.1E584Q hexamers in the presence of 3 mM
ATP. Number of hexamers observed = 10; total
observation period = 55.5 s. Red line, doubleGaussian fit result; blue lines, Gaussian components in double-Gaussian fit. Peak value
represents mean ± SD. Longer time courses
containing these two examples and additionaltime courses are shown in Figures S6A–S6D.
(D and E) Two representative 10 s time coursesof CDC-48.1E584Q hexamers in the presence of
3 mM ADP.
(F) Histogram of the angle of diametric lines of
CDC-48.1E584Q hexamers in the presence of 3 mM
ADP. Number of hexamers observed = 10; total
observation period = 83.5 s. Red line, doubleGaussian fit result; blue lines, Gaussian components in double-Gaussian fit. Peak value
represents mean ± SD. Longer time courses
containing these two examples and additional timecourses are shown in Figures S6E–S6H.
See also Figures S6I and S6J for histograms ofdwell time in the resting state and lifetime of therotated state, respectively.

and therefore senses the g phosphate of the bound ATP. Thissignaling may facilitate the binding of the fourth ATP. In mutant
CDC-48.1E584Q, the signaling is defective and the binding of
the fourth ATP may therefore be slow. The g phosphate sensing
by the Glu584 residue may be a key regulatory mechanism of therotational movement. For AAA+ proteins, the glutamate switch
has been proposed (Zhang and Wigley, 2008). The glutamate
switch regulates ATPase activity in response to the binding oftarget ligands by controlling the orientation of the catalytic Glu
residue, switching it between active and inactive conformations.
In the case of p97, the D2 domain, which is not directly involvedin substrate binding, interacts with the D1 domain through a region, where the glutamate switch exists, in the D2 domain. The
glutamate switch has also been identified in T7 DNA helicase,which is distinct from but related to AAA+ proteins (Satapathy
and Richardson, 2011). It has been revealed that the glutamate
switch of T7 DNA helicase couples dTTP hydrolysis to DNA binding, and that, when the catalytic Glu residue is replaced withglutamine, which is unable to sense the g phosphate, the alteredhelicase, unlike the wild-type helicase, binds DNA in the presence of dTDP. Therefore, it is reasonably assumed that the rotation of CDC-48.1E584Q may be induced by ADP as well as ATP bya similar mechanism. Further studies are needed to elucidate thedetail of this regulatory mechanism.
Recently, conformational changes of F1 ATPase have been
extensively investigated by high-speed AFM (Uchihashi et al.,2011). F1 ATPase is also a Walker-type hexameric ATPase of
the RecA fold as well as AAA+ family ATPases (Lupas and Martin,
2002). F1 is a motor in which three torque-generating b subunits
in the a3b3 stator ring sequentially undergo conformationalchanges upon ATP hydrolysis to rotate the central shaft g1998 Structure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reservedStructure
Rotation of p97 Analyzed by High-Speed AFM

Figure 7. Models for Nucleotide-Induced Rotation of CDC-48.1
Bottom views of CDC-48.1 hexamers and their predicted nucleotide states are schematically depicted. Models imply that ATP-binding to D2 induces conformational changes of the D2 domain, and the conformational changes of D2 result in twisting motion between the D2 and N-D1 rings. The D2 ring rotates 23 ± 8clockwise relative to the N-D1 ring when observed from the bottom. In the present high-speed AFM observation, however, the D2 ring is fixed on a surface of mica,
and therefore the D2 cannot be observed. Instead, the N-D1 side is observed, and the N-D1 ring rotates 23 ± 8 clockwise when observed from the top. The N-D1
ring and conformational changes in the D2 domain, except for the rotational movements of the D2 ring, are not drawn for simplicity. There are two states, resting
and rotated states. T represents ATP, and D represents ADP. The rotated state starts at state 2, where the third ATP binds to the D2 ring. Rate constants, which are
related to the lifetime of rotated state, were determined from the data of histogram of the lifetime of wild-type CDC-48.1 in the rotated position in the presence of
3 mM ATP (Figure S4G) for Model WT, and from that of CDC-48.1E584Q in the rotated position in the presence of 3 mM ATP (Figure S6J) for Model E584Q.
See also Table S1.

unidirectionally. It has been shown that the rotorless F1 still ‘‘rotates,’’ i.e., the three b subunits in the isolated a3b3 stator ring
cyclically propagate conformational states in the counterclockwise direction, similar to the rotary shaft rotation in F1 (Uchihashi
et al., 2011). Obviously, b subunits of F1 themselves undergo significant conformational changes during the cycle of ATP hydrolysis. Unfortunately, however, it is uncertain whether CDC-48.1
subunits undergo conformational changes during the cycle of
ATP hydrolysis, because the D2 ring of CDC-48.1 is fixed tothe surface of mica under the conditions used, and it is impossible to observe the D2 ring. Immobilization of the N-terminalside of CDC-48.1 to mica allows observation of the D2 ring.
Indeed, the N-terminally His-tagged CDC-48.1 could be immobilized through binding of His-tag to Ni-NTA-biotin as described,
but the resolution of captured images of the D2 ring has been
low. It is clear that the rotation of F1 ring is sequential and unidirectional under certain conditions, but the rotation of CDC-48.1
is a single-step back and forth in a twisting motion between
covalently connected two rings. Both F1 and CDC-48.1 are hexameric and structurally related, but modes of conformationalchanges seem to be significantly different. Each subunit of F1
has a single ATPase domain, whereas CDC-48.1 has two
ATPase domains in tandem.
Here, we establish the methodology to visualize structure andmovement of hexameric chaperones functioning in solution
simultaneously with high-speed AFM, which reveals nucleotide-induced conformational changes in a millisecond timescale. Our results provide insights into the molecular mechanismof the molecular chaperone, p97. In a future study, we would liketo improve procedures of immobilization of the N domain of
CDC-48.1 to a surface of mica to visualize the shape of the D2ring more clearly with high-speed AFM, which may allow us toobserve conformational changes of the D2 ring with a highATPase activity precisely.

EXPERIMENTAL PROCEDURES
Preparation of C. elegans CDC-48.1 Variants and Human VCP
Plasmids expressing His-tagged CDC-48.1 mutants and His-tagged human
VCP were constructed using the QuikChange kit (Stratagene). N-terminallyStructure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reserved 1999

Structure
Rotation of p97 Analyzed by High-Speed AFM

His-tagged wild-type and mutant CDC-48.1 proteins, and N-terminally Histagged human VCP were expressed in insect cells and purified as described
previously (Nishikori et al., 2011).

To determine the rate constants indicated in Model WT in Figure 7, the histograms of the lifetime of rotated state of wild-type CDC-48.1 in the presence of
3mM ATP (Figure S4G) were fitted by

ATPase Assay
ATPase activity was determined using the malachite green assay (Chan et al.,1986) as described previously (Nishikori et al., 2011).

nðtÞ = C

Fixation of CDC-48.1 and VCP onto Mica for AFM Observation
A droplet containing CDC-48.1 or VCP (10 nM) was deposited on a surface of
untreated mica for 10 min, followed by washing with buffer A (50 mM Tris-HCl[pH 8.0], 50 mM KCl, 30 mM b-mercaptoethanol, and 5 mM MgCl2). To preparea Ni-NTA coated mica, streptavidin two-dimensional (2D) crystals were formedon supported biotin-containing lipid bilayer as described previously (Yamamoto et al., 2009), and Ni-NTA-biotin was attached to streptavidin by depositing a droplet containing 0.1 mM Ni-NTA-biotin for 5 min. The sample stage
was mounted on the AFM head and immersed in buffer A with or without
ATP or ADP.
High-Speed AFM Apparatus
We used a home-built high-speed AFM apparatus (Ando et al., 2001; 2008).
The cantilevers (Olympus) were 6–7 mm long, 2 mm wide, and 90 nm thick.
Their spring constant was 0.1 Nm1, and their resonant frequency
and quality factor in water were 0.8–1 MHz and 2, respectively. For
AFM imaging, the free oscillation amplitude was 1 nm and the set-point
amplitude was around 90% of the free oscillation amplitude. The tapping
force estimated was less than 30 pN (Rodriguez and Garcia, 2003).
The probe tip was grown on the tip of a cantilever with electron beam deposition and was further sharpened by argon plasma etching (Wendel et al.,1995).
AFM Imaging and Image Processing
All AFM observations were performed at room temperature (24 C–26 C).
We usually used a scan area of 40 3 20 or 40 3 40 nm2 with 128 3 64 or
128 3 128 pixels. The AFM images were captured at frame rates of 10 fps.
A 3 3 3 pixel-average filter was applied to each tracked image to reduce noise.
Simulation of AFM Image
We used software (SPM simulator, Advanced Algorithm Systems, Tokyo,
Japan) to simulate AFM images of the p97 hexamer attached to a surfaceof mica. The simulation was carried out with a simple hard-sphere model as
described (Uchihashi et al., 2011). The cantilever tip was modeled as a circular cone (apex angle, 30 ) with a small sphere (radius, 8 nm) at the apex. Crystal structure of the apo p97 (Protein Data Bank [PDB] ID: 3CF3; Davies et al.,2008) was used as the sample. Each atom in the protein was modeled as a
hard sphere with a corresponding van der Waals radius. We simulated AFM
images using various radii for the tip-apex sphere and found that a radiusof 8 nm produced the images most similar to the actual AFM images. The
simulated images were processed by a low-pass filter with a cut-off wavelength of 2 nm, because the spatial resolution of the AFM image was approximately 2 nm judging from the 2D Fourier transformation of actual AFM
images.
Determination of Kinetic Parameters
Kinetic parameters were determined by fitting double exponentials to histograms using the software Origin 6.0 (Lightstone, Tokyo, Japan) as follows.
The histograms of the dwell time in the resting state and the lifetime ofrotated state of wild-type CDC-48.1 (Figure S4) and CDC-48.1E584Q (Figure S6) were fitted by the following equation to determine the preexponential
factors and time constants in each exponential term (see also the left columnof Table S1)





t
t
nðtÞ = A1 exp + A2 exp ;t1
t2(Equation 1)

where n(t) is the number of event that occurred during time interval betweent and t + dt.







1
k4 k2 + k3  1=t 2tk2 + k2 + k3 exp 
1=t1  1=t2
t 1 k3
t1






1
k4 k2 + k3  1=t 1t
exp + C k2 + k2 + k3 ;
1=t2  1=t 1
t2 k3t2(Equation 2)

where C is constant, and time constants t1 and t2 are given by

t1 =

 
qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ11k2 + k3 + k3 + k4 + ðk2 + k3  k3  k4 Þ2 + 4k3 k32(Equation 3)

and

 
qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ11k2 + k3 + k3 + k4  ðk2 + k3  k3  k4 Þ2 + 4k3 k3t2 =:2(Equation 4)
For Model E584Q, the rate constants are determined by fitting the following
equation to the histogram of the lifetime of rotated state of CDC-48.1E584Q in
the presence of 3mM ATP (Figure S6J)k2 + k3  1=t 2
texp 
1=t1  1=t 2
t1
k2 + k3  1=t 1t
exp + C k2;
1=t2  1=t 1
t2

nðtÞ = C k2(Equation 5)

where C is constant, and time constants t1 and t2 are given by

t1 =

 
qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ11k2 + k3 + k3 + ðk2 + k3 + k3 Þ2  4k2 k3
2(Equation 6)

and

t2 =

 
qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ1
12:k2 + k3 + k3  ðk2 + k3 + k3 Þ  4k2 k3
2(Equation 7)

Calculation of Kinetic Parameters
Independently of the determination of kinetic parameters using Equation 1,
preexponential factors, A1 and A2, and time constants, t1 and t2, can be obtained by calculation from rate constants indicated in Figure 7, as indicatedin the right column of Table S1. For Model WT, time constants, t1 and t2, aredescribed as Equation 3 and Equation 4, respectively, while preexponential
factors, A1 and A2, are given by





1
k4
k2 + k3  1=t2
A1 = C k2 + k2 + k3 
1=t 1  1=t2
t 1 k3(Equation 8)
and





1
k4
k2 + k3  1=t 1
A2 = C k2 + k2 + k3 :
1=t2  1=t 1
t 2 k3(Equation 9)
For Model E584Q (rate constant k4 = 0), time constants, t1 and t2, aredescribed as Equation 6 and Equation 7, respectively, while preexponential
factors, A1 and A2, are given by2000 Structure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reservedStructure
Rotation of p97 Analyzed by High-Speed AFMk2 + k3  1=t 2
1=t1  1=t 2(Equation 10)k2 + k3  1=t1:
1=t 2  1=t1

(Equation 11)

A1 = C k2

Chang, L.F., Chen, S., Liu, C.C., Pan, X., Jiang, J., Bai, X.C., Xie, X., Wang,
H.W., and Sui, S.F. (2012). Structural characterization of full-length NSF and
20S particles. Nat. Struct. Mol. Biol. 19, 268–275.

and

A2 = C k2

Chan, K.M., Delfert, D., and Junger, K.D. (1986). A direct colorimetric assay forCa2+ -stimulated ATPase activity. Anal. Biochem. 157, 375–380.

SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures, one table, and four movies
and can be found with this article online at http://dx.doi.org/10.1016/j.str.
2013.08.017.

ACKNOWLEDGMENTS
We thank A. Miyagi for setting up the high-speed AFM apparatus used in thisstudy, K. Karata for cloning of the human VCP gene, T. Uchihashi for simulationof AFM image, K. Namba for advice on image processing, and T. Yamaguchi
for suggestions to the presentation in enzymology. We are indebted to A. Förster, X. Zhang, and P. Freemont for stimulating discussion. We also thankmembers of the Ogura and Ando laboratories for stimulating discussion andsuggestions. This work was supported in part by the Core Research for Evolutional Science and Technology (CREST) program grant from the Japan
Science and Technology Agency (JST; to T.A. and T.O.); by grants from the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) of
Japan (19058012, 24370056, 24659432, and 25117517 to T.O.; 24117001a
and 24117004a to T.K.; and 20221006 to T.A.); by the program of Joint Usage/Research Center for Developmental Medicine, Institute of Molecular
Embryology and Genetics, Kumamoto University (to D.Y.); and by the Global
COE Program (Cell Fate Regulation Research and Education Unit) from
MEXT, Japan. K.N. and K.-i.A.-M. were research associate and juniorresearch associate, respectively, of the Global COE. T.O. was a Global COE
member.
Received: March 20, 2013
Revised: August 19, 2013
Accepted: August 19, 2013
Published: September 19, 2013

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Structure
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2002 Structure 21, 1992–2002, November 5, 2013 ª2013 Elsevier Ltd All rights reserved