Article

Chaperonin GroEL–GroES Functions as both
Alternating and Non-Alternating Engines

Daisuke Yamamoto and Toshio Ando
Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
CREST, JST, Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan

Correspondence to Toshio Ando: Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192,
Japan. tando@staff.kanazawa-u.ac.jp
http://dx.doi.org/10.1016/j.jmb.2016.06.017
Edited by J. Buchner

Abstract
A double ring-shaped GroEL consisting of 14 ATPase subunits assists protein folding, together withco-chaperonin GroES. The dynamic GroEL–GroES interaction is actively involved in the chaperonin reaction.
Therefore, revealing this dynamic interaction is a key to understanding the operation principle of GroEL.
Nevertheless, how this interaction proceeds in the reaction cycle has long been controversial. Here, we
directly imaged GroEL–GroES interaction in the presence of disulfide-reduced α-lactalbumin as a substrate
protein using high-speed atomic force microscopy. This real-time imaging revealed the occurrence of primary,symmetric GroEL:GroES2 and secondary, asymmetric GroEL:GroES1 complexes. Remarkably, the reaction
was observed to often branch into main and side pathways. In the main pathway, alternate binding andrelease of GroES occurs at the two rings, indicating tight cooperation between the two rings. In the sidepathway, however, this cooperation is disrupted, resulting in the interruption of alternating rhythm. Fromvarious properties observed for both pathways, we provide mechanistic insight into the alternate andnon-alternate operations of the two-engine system.
© 2016 Elsevier Ltd. All rights reserved.

Introduction
Chaperonins are a structurally conserved class ofmolecular chaperones that mediate protein folding to
the native, functional state in cells [1,2]. The beststudied chaperonin, Escherichia coli GroEL, is acylindrical protein complex formed by two heptamericrings stacked back to back, each consisting of identicalATPase subunits [3]. GroEL functions together with thelid-like co-chaperonin GroES. GroES is a single
homo-heptameric ring and binds to the ends of the
GroEL cylinder depending on the nucleotide state of
GroEL. The mechanism of productive protein folding
assisted by GroEL and GroES has been studied
extensively [4–6]. Nonnative, unfolded proteins with
exposed hydrophobic residues bind to GroEL at its
apical domain that presents a hydrophobic surface
for this binding [7]. Then, the substrate protein is
encapsulated into the hydrophilic cavity of GroEL
upon its binding to ATP and GroES, accompanied bya large conformational change of GroEL [8,9]. The
0022-2836/© 2016 Elsevier Ltd. All rights reserved.

encapsulated protein can fold in this environment,
which takes several seconds, the time needed for
one ATP turnover cycle to be completed in the
GroES-bound ring. Subsequently, GroES dissociates
and then the substrate protein is released from GroEL.
Because the two rings of GroEL are identical, theseprocesses proceed at each ring of GroEL.
However, how the reaction cycle proceeds in the“two-engine” system has long been controversial. In a
widely accepted model, it is postulated that only one
ring binds GroES throughout the cycle, so that theasymmetric GroEL:GroES1 complexes (referred to as
the bullet complexes) are exclusively formed in thesteady-state ATPase cycle. The origin of this asymmetry has been considered to be the inter-ring
negative cooperativity in ATP binding by GroEL [10–12]; that is, only one ring can bind ATP, resulting in the
exclusive formation of bullet complexes because
GroES can only bind to the ATP-bound ring [13],although GroES is thought to be able to bind to the
ADP–Pi-bound ring as well. Only after the bound ATP
J Mol Biol (2016) 428, 3090–3101

3091

Chaperonin GroEL–GroES reaction in two pathways

is hydrolyzed in the GroES-bound ring (cis-ring), theopposite GroES-free ring (trans-ring) can bind ATP.
Actual ATP binding to the trans-ring induces therelease of GroES, ADP, and the encapsulated
substrate protein from the opposite ring [14,15], while
the second GroES binds to the trans-ring to form a new
cis-ring. Thus, this model has concluded that the tworings of GroEL alternately bind and release GroES andhence alternately function as well. In another model,
however, both rings of GroEL are supposed to be able
to bind ATP simultaneously and hence also bind GroES
simultaneously to form symmetric GroEL:GroES2
complexes (referred to as the football complexes).
Several lines of evidence have been provided for the
existence of a large population of the football complexes in the presence of ATP [16–27]. However, thismodel has not gained broad consensus. This is mainly
because the methods used hardly allow direct detectionof dynamic molecular events occurring at each ring.
Here, using high-speed atomic force microscopy(HS-AFM) [28,29], we directly observed the dynamic
GroES association and dissociation events at each ringof individual GroEL molecules during the steady-state
ATPase cycle in the presence of disulfide-reduced
α-lactalbumin. HS-AFM is now established and has
recently been used with great success to visualize
protein molecules in dynamic action [30–32]. The
GroEL–GroES interaction was previously observedby HS-AFM during the course of establishment of thismicroscopy [33,34]. However, in these studies, GroEL
was immobilized in an end-up orientation on a mica
surface, making it infeasible to study the two-engine
cycle. In the present study, we used streptavidin
two-dimensional (2D) crystals as a substrate, onto
which GroEL molecules were immobilized in a side-onorientation through the streptavidin–biotin linkage [35].
This system allowed us to study the dynamic GroEL–
GroES interaction occurring during the two-engine
cycle at a nearly saturating concentration of GroES.
The HS-AFM imaging of the GroEL–GroES interactionin the steady-state ATPase cycle revealed various
properties of the interaction and thus provided mechanistic insight into the two-engine cycle as follows. The
symmetric football complexes are primarily formed,
while the two engines operate alternately in a main
pathway but non-alternately and non-simultaneously ina side pathway. The alternate operation in the main
pathway is made possible by inter-ring communications; ATP hydrolysis into ADP–Pi in one ring triggers
GroES dissociation from the opposite ring, while theresulting asymmetric bullet structure retards ADP
dissociation from the trans-ring. This retardation cancontribute to providing an enough time for the substrate
protein to be released from the trans-ring but in turn
could possibly result in frequent, incomplete nucleotide
replacement of ADP with ATP at the trans-ring. With
this incomplete exchange, the inter-ring communication is very likely to be vanished, and therefore, thereaction pathway is sidetracked into the side pathway.

Results
Patterns of dynamic GroEL–GroES interaction
For HS-AFM visualization of dynamic GroEL–
GroES interaction at the two rings of GroEL, theD490C GroEL mutant biotinylated at Cys490 was
immobilized in a side-on orientation on the streptavidin 2D crystal surface [35,36] (Fig. 1a). Since thissurface is highly resistant to nonspecific protein
binding [35], GroES appeared in HS-AFM images
only when it was bound to the immobilized GroEL.
This property allowed the use of a high concentrationof GroES (1 μM), unlike conventional single-molecule
fluorescence microscopy. Figure 1b presents
HS-AFM images that were captured at ~ 4 frames/s(fps) for dynamic GroEL–GroES interaction in thepresence of ATP and a substrate protein,
disulfide-reduced α-lactalbumin (Movie S1). The
successive images clearly displayed multiple roundsof GroES association/dissociation events at each ringof GroEL (Fig. 1b and c).
In the repeated cycles, the symmetric footballcomplexes appeared most frequently (~ 67%), while
the bullet complexes appeared moderately (~ 33%)
(Fig. 2A), consistent with a previous electron
microscopy study [37]. Next, we analyzed the orderof association and dissociation of GroES at the tworings by choosing the bullet complexes as an initialstate (Fig. 2B). These dynamic events observed arelargely classified into Type I and Type II; in Type I,
the cis/trans states interchange between the tworings after a round of association and dissociation of
GroES, resulting in the polarity change between theinitial and second bullet complexes; whereas in TypeII, no cis/trans interchange occurs, resulting in nochange of the polarity. The probabilities of occurrence of Type I and Type II processes are ~ 0.69 and~ 0.31, respectively. These processes mostly proceeded through formation of the football complexes(Fig. 2Bb and Be). To a lesser extent, no intermediate state appeared in Type I process (Fig. 2Bc). In
addition, processes going through the GroES-freestate were only rarely observed (Fig. 2Ba and Bd).
As described later, the occurrence of the two types ofprocesses, Type I and Type II, is not an artifact thatmight have resulted from miss-capture of the second
bullet complexes due to insufficient temporal resolution, but is consistent with a previous singlemolecule fluorescence microscopy study [21].
Decay kinetics of football and bullet complexes
The football complexes formed in Type I and Type IIprocesses are different species, as revealed by their
distinct decay kinetics. The histogram of lifetime forthe football complex in Type I process (hereafter werefer to as Type I football) was well fitted to a

3092

Chaperonin GroEL–GroES reaction in two pathways

Fig. 1. GroEL–GroES interaction observed by HS-AFM. (a) Schematic illustration of the assay system used for
HS-AFM imaging of GroEL–GroES interaction. Streptavidin was two-dimensionally crystallized on a mica-supported lipid
bilayer surface containing biotin-lipid. D490C GroEL biotinylated at Cys490 locating at its equatorial domain was
immobilized on the streptavidin 2D crystal surface through the biotin–streptavidin linkage. The bulk solution includes 1 μM
GroES, 1 μM denatured (disulfide-reduced) α-lactalbumin, and 2 mM ATP. (b) HS-AFM images captured at ~ 4 fps of
GroES binding to and dissociating from the GroEL rings. The dashed lines indicate the positions of toroid ends of the
GroEL molecule. The arrowheads indicate GroES bound to GroEL. Z-scale: 15 nm. (c) Time course of the association anddissociation of GroES at each ring of GroEL observed in (b).

single-exponential function with a rate constant ofχ 2 = 0.84, p N 0.71) (Fig. 3a, blue
k F-I = 0.49 s −1 ( ~lines), so that Type I football decays in the first-order
reaction. In contrast, the histogram of lifetime for the

football complex in Type II process (we refer to as
Type II football) showed a maximum at ~ 2 s and waswell fitted to a curve obtained for a sequential two-stepreaction with rate constants of k F-II1 = 1.14 s − 1 andChaperonin GroEL–GroES reaction in two pathways

3093

Fig. 2. Population of GroEL–GroES complexes and their dynamic appearance and disappearance observed by
HS-AFM. (A) Population of species in the presence of 2 mM ATP and 1 μM substrate protein (disulfide-reduced
α-lactalbumin). “n” indicates the total number of frames captured. (B) Patterns and relative proportion of the sequential
GroES binding and release events observed during the steady-state ATPase cycle. “n” indicates the total number of events
detected.

~2 = 1.46, p = 0.054) (Fig. 3b, blue
k F-II2 = 0.59 s −1 ( χlines; Note S1). This suggests that in Type II football,the dissociation of the second bound GroES is likely to
be caused by certain reactions occurring in its boundring with the rate constants k F-II1 and k F-II2, as the
decay process of Type II football is identical to aprocess that leads to dissociation of the second bound
GroES, unlike in Type I process where the decay ofType I football occurs by dissociation of the earlybound GroES.
Supposing that Type II football is apparently
formed due to miss-capture of a bullet complex that
occurs on route to the formation of the second
football complex, its lifetime should be approximately
2/k F-I = 4.08 s, and its decay kinetics should follow asequential two-step reaction with rate constants,
both of which are identical to k F-I = 0.49 s − 1 .

Because the decay kinetics exhibited by Type II
football is largely inconsistent with these features,Type II football is a real entity.
Next, we analyzed the decay kinetics of the bullet
complexes to obtain a clue to an origin of theformation of the two types of football complexes. The
lifetime of the bullet complex that was followed byType I football was well fitted to a single-exponential~2 =function with a rate constant of k B-I = 2.75 s − 1 ( χ
0.48, p N 0.94) (Fig. 3c, blue lines). Also, the lifetimeof the bullet complex that was followed by Type II
football was well fitted to a single-exponentialfunction, but its rate constant k B-II was noticeably~2 = 0.52,
smaller than k B-I, that is, k B-II = 2.02 s − 1 ( χp N 0.94) (Fig. 3d). Therefore, there are two types ofbullet complexes; one (we refer to as Type I bullet)
leads to the formation of Type I football, and the3094

Chaperonin GroEL–GroES reaction in two pathways

Fig. 3. Histograms and their best fitting results for lifetime of GroEL–GroES complexes and residence time of bound
GroES. The insets in (a–e) show the cumulated numbers of corresponding events (gray bars) together with curves
calculated using rate constants obtained by fitting their histograms to corresponding models (blue lines for a–d; blue, green
and red lines for e). “n” attached to each inset indicates the total number of observed events. (a) Histogram (gray bars) for
lifetime of Type I football and the best result of its fitting to a single-exponential function (blue line). (b) Histogram (graybars) for lifetime of Type II football and the best result of its fitting to a sequential two-step reaction model (blue line). (c)Histogram (gray bars) for lifetime of Type I bullet and the best result of its fitting to a single-exponential function (blue line).
(d) Histogram (gray bars) for lifetime of Type II bullet and the best result of its fitting to a single-exponential function (blueline). (e) Histogram (gray bars) for residence time of GroES and the best result of its fitting to a sequential four-step reaction
model (solid and dashed red lines). The dashed red lines show the best result of fitting performed under the restriction of
k3 = k B-I and k4 = k F-1, while the solid red lines show the best result of fitting performed without restriction. The green andblues lines show the best results obtained when the histogram for residence time of GroES was fitted to sequential
two-step and three-step reaction models, respectively. The inset (right) shows the initial lag-time phase of the cumulated
number of GroES dissociation events.

other bullet (we refer to as Type II bullet) leads to theformation of Type II football. However, the ratio of therate constants k B-I/k B-II ≈ 1.36 cannot account forthe probabilities of occurrence of Type I and Type II
footballs (i.e., ~ 0.67 and ~ 0.33, respectively), as

described below. The rate constants, k F-I = 0.49 s − 1and k B-I = 2.75 s − 1, provide the probabilities ofappearance of Type I football and Type I bullet as0.85 and 0.15, respectively, which are apparently
inconsistent with those mentioned above, even

Chaperonin GroEL–GroES reaction in two pathways

when considering the presence of Type II process.
This discrepancy is due to the limited time ofobservation of the reaction cycle (Note S2).
Type I football could possibly be classified into two
subtypes responsible for the formation of Type I andType II bullet complexes, but it is not the case. This isbecause the rate of first-order transition from Type I
football to Type II bullet was nearly identical to that ofType I football to Type I bullet (Fig. S1). Thisconclusion was also supported by the fact that bothType I and Type II bullets are formed after Type II
football, with respective probabilities similar to those of
the occurrence of Type I and Type II bullets after TypeI football (probabilities: 449/671 ≈ 0.67 for Type I
bullet formation, and 222/671 ≈ 0.33 for Type II bullet
formation). In fact, the numbers of events that Type IIprocess occurred by n-times in succession were 67(N2 = 172), 19 (N3 = 53), and 4 (N4 = 15), where Nn(n = 2–4) represents the total number of Type I andType II bullet complexes formed after Type II footballin the (n − 1)th round. Thus, the probability of going
through Type II process is approximately kept
constant at ~ 0.33, regardless of the number ofsuccessive rounds of Type II process. As such, thetwo types of bullet complexes always occur withrespective constant probabilities, after either type offootball complex.
Kinetic reaction scheme
From the results above and the analyses described
below, the reaction scheme for GroEL–GroES

3095
interaction in the steady-state ATPase cycle wasconstructed (Fig. 4). The reaction cycle proceeds
through two distinct main and side pathways, where
Type I and Type II footballs are formed, respectively.
Branching into the two pathways occurs at and isdetermined by the bullet complexes. The main
pathway is consistent with a symmetric chaperonin
cycle as proposed previously [24]. In contrast, in theside pathway, the product formed by the decay ofType II football is in the same cis/trans-ringarrangement as that of the previous bullet complex;that is, the GroES that has been bound until thereaction reaches the branching point is kept
attached during the entire period of the sidepathway. Therefore, its residence time, ~ 7.5 s (orlonger when the reaction proceeds in succession to
the side pathway), is significantly longer than that in
the main pathway, ~ 4.4 s. On contrary, the residence
time of the second bound GroES (~ 2.6 s) in the sidepathway, which is identical to the lifetime of Type II
football, is significantly shorter than the residence
times of the early bound GroES (N ~ 7.5 s) and thebound GroES in the main cycle (~ 4.4 s).
From the values obtained above for k F-I, k B-I, k F-II 1,
F-II
k 2, and k B-II and from the probability of occurrenceof Type II bullet, r ≈ 1/3, we obtained the average
cycle time of GroEL–GroES interaction, b Tc N ≈6.33 s, in a way described in Note S3. Supposingthat 14 ATP molecules are hydrolyzed per GroEL
molecule during b Tc N, the steady-state ATPase
activity is estimated to be 2.2 s − 1 per GroEL
molecule. This estimated value is somewhat larger

Fig. 4. Kinetic reaction scheme of GroEL–GroES interaction revealed by HS-AFM imaging. The football complexes
shown in pale colors are kinetically distinct from the respective football complexes initially formed upon GroES binding.
The solid black arrows indicate reactions in the main circular pathway, whereas the solid green arrows indicate those in theside pathway. The dashed red arrows indicate reaction processes estimated from the residence time of bound GroES. The
order of k F − II1 and k F − II2 was assigned as shown here, considering the fact that the value of k F − II2 = 0.59 s − 1 is smaller
than the smallest value reported for the rate of substrate encapsulation reaction that occurs after GroES binding to thesame ring of GroEL. The order of k1 and k2 was assigned as shown here, considering the fact that the value of k1 is
identical to the value of k F − II1. In the side pathway, the coexistence of ATP and ADP in one ring is shown but hypothetical.

3096
than but similar to the value of 1.5 s − 1 per GroEL
molecule measured biochemically for GroEL–GroESin the presence of an unfoldable substrate protein,α-lactalbumin [38], the same substrate protein usedin the present study.
Type II football in the side pathway shown in palecolors (Fig. 4) indicates an intermediate state that is
distinct from Type II football initially formed upon
GroES binding. The existence of this intermediate
state was deduced from the histogram for lifetime ofType II football as mentioned above (Fig. 3b). Type I
football shown in pale colors (Fig. 4) is discussed in
the next section.
Kinetics undergone by bound GroES in themain pathway
Previous single-molecule fluorescence microcopy
measurements have shown that dissociation of
GroES from a GroEL ring occurs in two steps,
through the formation of one kinetic intermediate[36,39]. In these studies, however, measurements
were performed under the condition that only one
GroES was bound to GroEL (i.e., at a low concentration of GroES, 4 nM). Here, we examined the
kinetics undergone by a bound GroES in the main
circular pathway under the condition that the footballcomplexes are predominantly formed in the presence of 1 μM GroES. Figure 3e shows a histogram
for the residence time of GroES. As was the casewith the previous studies, the distribution of theresidence time showed a maximum, but it significantly deviated from the curve best fitted to a
~2 = 1.51, p = 0.016;
sequential two-step reaction ( χFig. 3e, green lines). For details of the residence
time analysis, see Notes S4 and S5. It would be
most plausible that before the final dissociation, thebound GroES simply undergoes the first footballcomplex, the subsequent bullet complex and thesecond football complex in this order. However, thehistogram largely deviates from the corresponding
curve (Fig. S2). A deviation was also noticed,
although to a lesser extent, even when the histogram was fitted to a sequential three-step reaction~2 = 0.92, p = 0.60), as depictedwithout restriction ( χin the cumulated number of GroES dissociation
events (Fig. 3e, blue lines). In this fitting (Fig. 3e,right, blue line), a significant advancement is evident
at the initial phase in the lag period, compared to theexperimental data (Fig. 3e, right, black dots).
Therefore, we postulate that the bound GroES
undergoes three intermediates (besides the initialfootball complex) before its dissociation. The histogram was well fitted to a curve for a sequential
four-step reaction with rate constants of k1 =0.92 s − 1 , k2 = 0.90 s − 1 , k3 = 2.81 s − 1 , and k4 =~2 = 0.70, p = 0.90) (Fig. 3e, solid red
0.51 s − 1 ( χlines). The cycle time of the main pathway calculated from the values of these four rate constants andChaperonin GroEL–GroES reaction in two pathways

k B-I becomes 4.89 s, which is very close to that
calculated from the values of k B-I and k F-I , that is,
2 × (1/k F-I + 1/k B-I ) ~ 4.80 s.
However, precisely determining the four rate
constants from one histogram is difficult. In addition,
the histogram does not tell the order of reactions
corresponding to these four rate constants. Therefore, we constructed a sequential four-step reaction
model by considering several issues and reassessing the histogram under plausible restrictions, as
described below. First, the values of k3 = 2.81 s − 1and k4 = 0.51 s − 1 are similar to the values of k B-I =2.75 s − 1 and k F-I = 0.49 s − 1, respectively, indicatingthat the reaction step corresponding to k3 occurs at
decay of the bullet complex, while the reaction stepcorresponding to k4 occurs at decay of either the
initial or final football. Moreover, the sum of thevalues of 1/k1 and 1/k2, ~ 2.2 s approximately
coincides with 1/k F-I (~ 2.04 s). It is well known that
substrate protein is encapsulated into the internal
cavity of GroEL after the binding of ATP and GroES
to the trans-ring, which instantly induces movementof the apical domain of the newly formed cis-ring.
This apical domain movement has been reported tooccur in 0.56–1.47 s (rate constant, 0.68–1.8 s − 1)
after ATP addition, depending on the particular
substrate protein [40]. Since the values of k1 andk2 are in this range, either k1 or k2 is very likely torepresent the rate of encapsulation. Therefore, two
successive reaction steps corresponding to k1 andk2 occur during the decay of the initial footballcomplex (see Fig. 4), and the rate constant k4 cannow be assigned to the rate of final dissociation of
the second bound GroES (at the decay of the last
football complex). However, the dissociation ofanother (i.e., early bound) GroES from the initialfootball occurs in one step, suggesting that this
GroES dissociation occurs in parallel to the encapsulation reaction and the subsequent unspecified
reaction (corresponding to either k1 or k2) occurringin the opposite ring. Because of 1/k1 + 1/k2 ≈ 1/k F-I,
completion of this unspecified reaction must be
synchronized with the dissociation of the earlybound GroES. Therefore, the encapsulation reaction
and the apical domain movement do not seem toaffect the counter ring. Collectively, we conclude
that after GroES binds to a GroEL ring, this GroES
undergoes the football complex (depicted inpale colors in Fig. 4), the subsequent bullet complex
and the last football complex in this order. Following
this scheme, we reassessed the histogram forthe residence time of GroES under the restrictionof 1/k1 + 1/k 2 = 1/k F-I , k3 = k B-I , and k4 = k F-I ,~2 =resulting in k1 = 1.14 s − 1 and k2 = 0.87 s − 1 ( χ
0.73, p = 0.88). The fitting curve obtained by thisreassessment (Fig. 3e, dashed red lines) was nearly
indistinguishable from the initial one (Fig. 3e, solid
red lines). Note that k1 = 1.14 s − 1 is identical to thevalue of k F-II 1.

Chaperonin GroEL–GroES reaction in two pathways

Discussion
The acquired HS-AFM images of GroEL–GroES
interaction in the presence of α-lactalbumin indisputably displayed that the football complexes areindeed primarily formed during the repeated reaction
cycles. Moreover, the HS-AFM images showed thatin the main pathway, the two rings of GroEL operatealternately as previously postulated [21,24,25].
Cooperative interactions between the two rings
must govern this rhythmic, alternate operation. In aprevailing view [2,4], a negative, cooperative effectbetween the two rings has been considered to inhibit
ATP binding to the trans-ring until one ATP turnoveris completed in the cis-ring, resulting in exclusive
formation of the bullet complexes in the reaction
cycle. Our results are inconsistent with this view.
However, this prevailing view has now been somewhat modified by a recent fluorescence crosscorrelation spectroscopy study [38]. This study
showed that symmetric and asymmetric complexes
are formed by 54% (close to our observation of 67%)
and 23%, respectively, in the presence of unfoldable
substrate proteins α-lactalbumin and α-casein,
whereas in the presence of foldable substrate
proteins, symmetric complexes are formed by lessthan 10% [38]. In the near future, this dependence
on substrate proteins should be further assessedusing different methods including HS-AFM because
the formation of football complexes with populations
much larger than 10% has been reported even in thepresence of foldable substrate proteins [17–23,26,27]. In another model, although in the caseof the absence of substrate protein, ADP dissociation from the trans-ring has been considered to limit
the reaction cycle, resulting in the accumulation ofbullet complexes. In fact, when ATP and ADP coexist
in solution, ADP has been shown to be bound to thetrans-ring [20,41]. Moreover, it has been shown thateven after the detachment of GroES from the cis-ring,
ADP resides in the same ring [42,43]. However, in a
related model, this negative cooperativity effect on the
ADP release from the trans-ring has been considered
to be weakened by substrate protein bound to thesame ring. Therefore, binding of ATP and GroES to thetrans-ring is accelerated [24,41], and hence, thefootball complex is considered to be formed during
the chaperonin cycle [22,25].
However, as shown in our HS-AFM observation,
Type I bullet stays for 1/k B-I ~ 0.36 s even in thepresence of substrate protein and 1 μM GroES. This
lifetime is much longer than the time required for
GroES binding to the ATP-bound trans-ring, considering the second-order rate constant for GroES
binding in the presence of substrate protein, 1–
3 × 10 7 M − 1 s − 1 [40] and the GroES concentration
used here. As such, regarding ADP dissociation,negative cooperativity still effectively acts on thetrans-ring even in the presence of substrate protein.

3097
This suppression of ADP release in the asymmetricbullet complex is reminiscent of the hand-over-handmovement of myosin V on actin; its identical twoheads alternately take the leading and trailing
positions. This alternate process is made possible
by strain-mediated suppression of ADP release fromthe leading head [30,44]. Therefore, suppression of
ADP release in an asymmetric structure seems to be
a common strategy for alternate operation of
two-engine ATPase systems.
In the main pathway (see Fig. 4), after binding to
the trans-ring of GroEL, the bound GroES undergoes
three intermediates before dissociation (besides theinitial football complex itself), as revealed by its
residence time analysis. Two of the three intermediates are Type I bullet and the last football complex.
This coincidence of the intermediate species indicates that the two rings communicate with eachother in these two intermediate states. In Type I
bullet, a negative cooperativity effect exists, asmentioned above. In the final football complex, apositive cooperativity effect that induces the final
dissociation of the GroES must also exist . In orderfor this positive cooperative effect to engender, acertain reaction must have proceeded in the
opposite ring until or just before the final dissociationof the bound GroES. During this period, encapsulation of substrate protein into the cavity occurs in the
new cis-ring but does not seem to affect the opposite
ring because the encapsulation occurs earlier than
the final dissociation of GroES and because thisdissociation occurs in one step. The rate of ATPhydrolysis to ADP–Pi in the presence of foldable
substrate proteins has been reported to be in therange of 0.31 s − 1 − 0.36 s − 1 [39,45]. Taking into
account the higher ATPase activities of GroEL in thepresence of unfoldable substrate proteins [38], thevalue of k F-I (0.49 s − 1 ) can be considered tocorrespond to the rate of ATP hydrolysis into ADP–
Pi. Therefore, we propose that ATP hydrolysis in the
new cis-ring triggers Pi release and hence final
dissociation of GroES from the opposite ring.
However, this proposition appears inconsistent witha previous view that the binding of ATP to the transring (not ATP hydrolysis) triggers GroES dissociationfrom the cis ring, or the binding of both ATP andsubstrate protein synnergistically triggers it [14,15].
Nonetheless, this view has been gained for thechaperonin cycle where bullet complexes are
predominantly or exclusively formed in the presenceof foldable substrate proteins. Therefore, theinter-ring coupling for GroES dissociation may
possibly depend on the type of substrate proteins.
To understand the cause of branching into the sidepathway, we summarize its major properties: (i) The
early bound GroES never dissociates; rather, thenewly bound GroES dissociates at the exit of theside pathway; (ii) The formation of Type II bullet
leads to the side pathway; (iii) The bullet complex

3098
formed at the exit of the side pathway can proceed toeither pathway; (iv) The probabilities of branching
into the main and side pathways (~ 2/3 and ~ 1/3,
respectively) cannot be accounted for by the rate
constants k B-I = 2.75 s − 1 and k B-II = 2.02 s − 1 ;(v) The rate of Type II football formation (2.02 s − 1)is smaller than that of Type I football formation(2.75 s − 1); (vi) Type II football decays in two steps,
whereas Type I football decays in one step;(vii) Type II football has a moderately longer lifetime(~ 2.6 s) than Type I football (~ 2.04 s); and(viii) Importantly, the side pathway occurs even in
the absence of substrate protein (Fig. 5).
It has been postulated that the substrate protein
initially tethered to the apical domain of GroEL would
have two or three different fates [46,47]. However,these fates have nothing to do with the pathway
branching (Note S6) because the side pathway
occurs even in the absence of substrate protein.
Although there is no direct evidence at this stage, we
consider that the incomplete exchange of nucleotide
at the trans-ring may cause sidetracking into the sidepathway, while complete exchange of seven ADPs
with seven ATPs assures the GroE system to go
through the main pathway. In bullet complexes, therate of ADP dissociation from the trans-ring is
suppressed, as described above. This suppression
provides an enough time for the substrate protein to
be released from the trans-ring but in turn could

Chaperonin GroEL–GroES reaction in two pathways

possibly cause frequent, incomplete nucleotide
replacement at the trans-ring, which would direct
the reaction process toward the side pathway. Thishypothesis is consistent with partial stochasticity of
ATP hydrolysis, as suggested by a previous study[25], and with all the observed properties of Type IIprocess. For example, the partially remained ADP
should somewhat reduce the affinity of the trans-ringfor GroES, which is consistent with the smaller rate
constant k B-II = 2.02 s − 1 than k B-I = 2.75 s − 1. This
weaker GroEL–GroES association would possibly
reduce the rate of ATP hydrolysis to ADP–Pi in the
new cis-ring, consistent with the longer lifetime ofType II football than Type I football. Moreover, thehydrolysis of reduced number of ATP molecules in
the new cis-ring must significantly reduce its positive
cooperative effect on the dissociation of the earlybound GroES from the opposite ring, which isconsistent with the fact that in the side pathway,
the early bound GroES never dissociates.

Materials and Methods
Proteins
The D490C GroEL was produced by site-directedmutagenesis. D490C GroEL and wild-type GroES wereexpressed in E. coli XL1-Blue and purified as described

Fig. 5. Successive HS-AFM images showing dynamic GroEL–GroES interaction in the absence of substrate protein.
The numbers shown are the frame number. As indicated at frame 102, the polarity of the bullet complex is unchanged fromthe previous bullet (frame 89) after a round of dissociation and association of GroES. Although not shown in this figure, 7
out of 47 total GroES binding and release events observed showed the formation of Type II football. The imaging was
performed 22 °C in a solution containing 25 mM Hepes-KOH, 100 mM KCl and 5 mM MgCl2, 1 μM GroES, 2 mM ATP,and 2 mM DTT. Imaging rate, ~ 4 fps; imaging area, 95 × 41 nm 2.

3099

Chaperonin GroEL–GroES reaction in two pathways
previously [48]. Purified D490C GroEL was labeled withbiotin by the reaction with biotin-PEAC5-maleimide for
30 min at 25 °C as described [35]. The molar ratio of biotin
introduced per GroEL subunit was determined to be 0.8
using 4′-hydroxyazobenzene-2-carboxylic acid (Wako
Chemicals, Osaka) [49]. Streptavidin and α-lactalbumin
were purchased (Wako Chemicals, Osaka and Nacalai
Tesque, Kyoto, respectively).
Streptavidin 2D crystals
Streptavidin 2D crystals were prepared on the surface of
mica-supported lipid planar bilayer containing biotin-lipid,
as described [35]. Briefly, the mica-supported lipid bilayer
was first obtained by a vesicle fusion method. Afterwashing the excess lipids, crystallization of streptavidin
was performed by deposition of streptavidin (0.2 mg/ml)
dissolved in crystallization buffer [10 mM Hepes, 150 mM
NaCl, and 2 mM CaCl2 (pH 7.4)] on the lipid bilayer surface,followed by incubation for 2 h. Then, the streptavidin 2D
crystals were chemically stabilized by the application of
10 mM glutaraldehyde mixed with the crystallization buffer.
After 5 min of incubation, the reaction was quenched using
20 mM Tris added to the crystallization buffer.

inspection of the fitting results than the use of curves forlifetime or residence time distribution. The details of data
analysis for sequential three- and four-step reactions aredescribed in Note S5.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jmb.2016.06.017.

Acknowledgments
We thank Prof. Masasuke Yoshida for the criticalreading of the draft and for valuable comments andDr. Masaaki Taniguchi for technical assistance. This
work was supported by grants to T.A.: KAKENHI,
Grants-in-Aid for Basic Research S (#20221006 and#24227005), and Research on Innovative Area (Research in a proposed research area; #26119003) fromJapan Society for the Promotion of Science, andCREST program of Japan Science and Technology
Agency.
Received 9 March 2016;
Received in revised form 23 June 2016;
Accepted 27 June 2016
Available online 5 July 2016

HS-AFM
Observations were carried out in amplitude modulation
mode using a laboratory-built HS-AFM setup [28,29]. Smallcantilevers used are custom made by Olympus (spring
constant of 0.1 N/m and the first resonant frequency of
0.8 MHz in water). Sharp tips were fabricated on the original
tip by electron beam deposition and then by argon-plasma
etching. The biotinylated D490C GroEL diluted to 25 nM
was applied to the streptavidin 2D crystals. After 3 min of
incubation, unattached GroEL was washed out with buffer.
HS-AFM imaging was performed at an imaging rate of ~4 fps
at 22 °C in a solution containing 25 mM Hepes-KOH,
100 mM KCl and 5 mM MgCl2, 1 μM GroES, 1 μM bovine
α-lactalbumin, 2 mM ATP, and 2 mM DTT. For HS-AFM
imaging in the absence of substrate protein shown in Fig. 5,bovine α-lactalbumin was omitted from the solution.

Keywords:
GroEL–GroES interaction;
high-speed AFM;
cooperativity;
chaperonin
Present address: D. Yamamoto, Department of Applied
Physics, Fukuoka University, Nakakuma, Jyonan-ku,
Fukuoka 814-0180, Japan.
Abbreviations used:
HS-AFM, high-speed atomic force microscopy; 2D,
two-dimensional.

Data analyses
The species of GroEL–GroES complexes were able tobe identified by visual inspection of HS-AFM images,
thanks to the high-resolution images. The analyses oflifetimes of the bullet and football complexes and theresidence time of bound GroES were performed with asoftware program constructed using Mathematica 10.2
(Wolfram Research, Illinois). The histograms for lifetime ofType I football and Type I and II bullets were fitted tosingle-exponential decay functions. The histogram forlifetime of Type II football was fitted to an equation for asequential two-step reaction (Note S1). The residence
time of GroES in the main pathway was fitted to equationsof sequential two-step, three-step, or four-step reactions(Note S4). The fitting results were also depicted withcurves obtained by using corresponding equations for the
cumulated number of events that occur during the period
from Δt to nΔt, where n is the integer and Δt is the frame
time of imaging (Note S4). This depiction provides better

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