Specific cleavage of the DNase-I binding loop dramatically decreases the thermal stability of actin

Specific cleavage of the DNase-I binding loop dramaticallydecreases the thermal stability of actinAnastasia V. Pivovarova1, Sofia Yu. Khaitlina2 and Dmitrii I. Levitsky1,3

1 A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia

2 Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia

3 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia

Introduction

Actin is one of the most abundant and highly conserved

cell proteins. It is involved in many different cellular

processes that are essential for growth, differentiation

and motility. Actin is found in two main states: as

monomers (G-actin) and as a helical polymer (F-actin).

Polymerization of G-actin into F-actin is accompanied

by hydrolysis of tightly bound ATP followed by a

slower release of Pi; as a result, protomers of F-actin

contain tightly bound ATP, ADP or ADP-Pi.

The atomic resolution structures of G-actin revealed

that it is divided into two easily distinguishable

domains by a deep cleft containing the tightly bound

nucleotide and cation [1]. The nucleotide-binding cleft

was suggested to exist in two main states, closed and

open [2,3], and solution studies on nucleotide exchange

and susceptibility of the cleft to limited proteolysis

appear to be consistent with the opening of the cleft

upon the transition from the ATP- to ADP-G-actin

Keywords

actin; differential scanning calorimetry;

DNase-I binding loop; proteolytic cleavage;

thermal unfolding

Correspondence

D. I. Levitsky, A. N. Bach Institute of

Biochemistry, Russian Academy of

Sciences, Leninsky Prospect 33, 119071

Moscow, Russia

Fax: +7 495 954 2732;

Tel: +7 495 952 1384

E-mail: [email protected].

(Received 10 June 2010, revised 14 July

2010, accepted 16 July 2010)

doi:10.1111/j.1742-4658.2010.07782.x

Differential scanning calorimetry was used to investigate the thermal

unfolding of actin specifically cleaved within the DNaseI-binding loop

between residues Met47-Gly48 or Gly42-Val43 by two bacterial proteases,

subtilisin or ECP32 ⁄ grimelysin (ECP), respectively. The results obtained

show that both cleavages strongly decreased the thermal stability of mono-

meric actin with either ATP or ADP as a bound nucleotide. An even more

pronounced difference in the thermal stability between the cleaved and

intact actin was observed when both actins were polymerized into fila-

ments. Similar to intact F-actin, both cleaved F-actins were significantly

stabilized by phalloidin and aluminum fluoride; however, in all cases, the

thermal stability of the cleaved F-actins was much lower than that of intact

F-actin, and the stability of ECP-cleaved F-actin was lower than that of

subtilisin-cleaved F-actin. These results confirm that the DNaseI-binding

loop is involved in the stabilization of the actin structure, both in mono-

mers and in the filament subunits, and suggest that the thermal stability of

actin depends, at least partially, on the conformation of the nucleotide-

binding cleft. Moreover, an additional destabilization of the unstable

cleaved actin upon ATP ⁄ADP replacement provides experimental evidence

for the highly dynamic actin structure that cannot be simply open or

closed, but rather should be considered as being able to adopt multiple

conformations.

Structured digital abstractl MINT-7980274: Actin (uniprotkb:P68135) and Actin (uniprotkb:P68135) bind (MI:0407) by

biophysical (MI:0013)

Abbreviations

D-loop, DNase I-binding loop; DSC, differential scanning calorimetry; DHcal, calorimetric enthalpy; Tm, thermal transition temperature.

3812 FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS

[4–6]. By contrast, in the numerous crystal structures

published to date, including those of ADP-G-actin

[7,8], the open nucleotide-binding cleft has been

observed only in profilin-bound actin crystals [9]. Only

the closed conformation was also revealed by molecu-

lar dynamics simulations of the crystal actin structures

[10–12], whereas metadynamics simulations have dem-

onstrated that the closed conformation of the nucleo-

tide-binding cleft is the most stable state only when

ATP is bound, and the ADP-bound state favors a

more open conformation of the cleft [13]. It is possible

therefore that crystallization favors a closed state for

G-actin even though the state of the cleft in solution

may vary [14,15].

The other nucleotide-dependent conformational

transitions in actin crystals involve so-called DNase

I-binding loop (D-loop) in subdomain 2 of the actin

molecule [7]. In tetramethylrhodamine-modified ADP-

G-actin, the D-loop was found to be in an a-helicalconformation, whereas this region was disordered in

the ATP-bound actin, suggesting that the D-loop folds

on ATP hydrolysis [7,16]. This transition within the

D-loop was supported by the results of metadynamic

simulations demonstrating a distinct allosteric relation-

ship between the conformation of the D-loop (ordered

or disordered) and the state of the nucleotide-binding

cleft (open or closed) [13]. These data are consistent

with the results of the biochemical observations indi-

cating that proteolytic modifications of the D-loop

affect the state of the interdomain cleft [5,17].

The D-loop of actin can be specifically cleaved with

two bacterial proteases. One of them is subtilisin,

which cleaves the D-loop between Met47 and Gly48

[18]. The other protease, which specifically cleaves

actin at the only site between Gly42 and Val43

(Fig. 1), was initially isolated and characterized as a

minor protein of lactose-negative Escherichia coli A2

strain and referred to as protease ECP32 [19,20]. More

recently, it was found that the A2 strain producing

protease ECP32 is identical to Serratia grimesii, and

therefore this enzyme was named grimelysin [21].

Although ECP32 and grimelysin were suggested to be

identical enzymes [21], both names are used in the lit-

erature. In accordance with previous studies [5,17], we

refer to this protease as ECP in the present study. The

nucleotide-binding cleft in both subtilisin-cleaved and

ECP-cleaved G-actin is clearly in a more open confor-

mation compared to intact actin, as demonstrated by

the increased nucleotide exchange rate in solution

[17,22] and their higher susceptibility to limited prote-

olysis [5,17]. By contrast, the crystal structure of ECP-

cleaved G-actin showed the nucleotide-binding cleft to

be in a typical closed conformation, probably as a

result of crystallization preferentially trapping actin in

only one of its possible conformations [14].

Taking into account the ambiguity of the nucleo-

tide-binding cleft conformation and its relationship

with the D-loop, the present study aimed to determine

whether the specific cleavage of the D-loop affects the

structural properties of the entire actin molecule and,

in particular, conformational transitions of the nucleo-

tide-binding cleft. For this purpose, we studied the

effects of the D-loop cleavage on the thermal unfold-

ing of G- and F-actin. Previously, the thermal unfold-

ing of G-actin containing different nucleotides was

indirectly studied with the DNase-I inhibition assay

[23] and by monitoring the change in absorbance of

tetramethylrhodamine-actin [24]. The results obtained

showed that replacement of the tightly bound ATP by

ADP led to a significant decrease in the thermal stabil-

ity of G-actin [23,24]. In the present study, we applied

differential scanning calorimetry (DSC), which is the

most direct and effective method for studying the ther-

mal unfolding of proteins. Previous studies have shown

that DSC can be successfully used to reveal the

changes in the thermal unfolding of actin induced by

interaction of G-actin with actin-binding proteins [25–

27], G–F transformation of actin, and stabilization of

F-actin by phalloidin and Pi analogs [28]. Moreover,

the effects of nucleotides on the thermal unfolding of

F-actin have been studied by this method and a ‘disso-

ciative’ mechanism for the thermal denaturation of

F-actin has been proposed [28,29].

In the present study, we used DSC to characterize

the thermal unfolding of actin specifically cleaved

within the D-loop by ECP or subtilisin. The results

obtained show for the first time that the cleavage

strongly decreases the thermal stability of G-actin and

especially that of F-actin, both in the absence and the

Fig. 1. 3D atomic structure of G-actin. The four subdomains are

indicated by the encircled numbers. Arrows show the cleavage

sites in the D-loop, between Gly42 and Val43 by ECP32 ⁄ grimelysin,

and between Met47 and Gly48 by subtilisin.

A. V. Pivovarova et al. Thermal unfolding of cleaved actin

FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS 3813

presence of phalloidin and Pi analogs. These results

are discussed with regard to a more open conforma-

tion of the interdomain cleft in the cleaved actin com-

pared to intact actin, both in the monomers and in the

filament subunits.

Results

Effects of the D-loop cleavage on the thermal

unfolding of G-actin

The excess heat capacity curves obtained for intact,

ECP-cleaved, and subtilisin-cleaved ATP-Ca-G-actins

are presented in Fig. 2. It is seen that the G-actin spe-

cies cleaved within the D-loop are clearly less thermo-

stable than noncleaved G-actin. The thermal

transitions of both ECP-cleaved and subtilisin-cleaved

G-actin are shifted to a lower temperature, by 4–5 �C,compared to that of intact G-actin, and the values

of calorimetric enthalpy, DHcal, determined for the

cleaved G-actins are � 57–63% of those for nonc-

leaved G-actin (Table 1). Thus, both cleavages within

the D-loop strongly decrease the thermal stability of

ATP-Ca-G-actin, with no significant difference

between the effects of ECP and subtilisin. It is

important to note that heating cleaved G-actins in the

calorimeter cell did not lead to any further proteolysis

of the proteins (Fig. 2, inset).

We also compared the thermal unfolding of intact

and ECP-cleaved G-actins in the different states, with

the tightly bound Ca2+ replaced by Mg2+ and with

the tightly bound ATP replaced by ADP (Fig. 3). The

replacement of Ca2+ by Mg2+ in ATP-G-actin had no

appreciable effect on the thermal unfolding of either

intact or ECP-cleaved G-actin: in both cases, it only

slightly decreased the maximum thermal transition

temperature (Tm), by 1–2 �C, with no effect on the

DHcal value (Table 1).

By contrast, the replacement of bound ATP by

ADP caused a dramatic decrease in the thermal stabil-

ity of G-actin. Intact ADP-Mg-G-actin demonstrated

the thermal transition with Tm of 48.8 �C (Fig. 3A)

(i.e. 11 �C less than that of ATP-Mg-G-actin) and its

calorimetric enthalpy (340 kJÆmol)1) was much

less than that of ATP-Mg-G-actin (570 kJÆmol)1)

(Table 1). Figure 3A shows that the sample contains

only ADP-actin because no peak at 60 �C (correspond-

ing to the thermal transition of ATP-Mg-actin) was

seen on the thermogram. A similar effect was observed

on ECP-cleaved Mg-G-actin with ATP replaced by

ADP (Fig. 3B). In this case, the nucleotide replace-

ment decreased the Tm by 9 �C and led to a more than

two-fold decrease in the DHcal value (Table 1).

Thermal unfolding of F-actin with the cleaved

D-loop

Previous studies have shown that ECP-cleaved actin is

unable to polymerize unless its tightly bound Ca2+ is

replaced with Mg2+, and that the Mg2+-bound form

has higher critical concentration and polymerizes more

slowly than Mg-G-actin cleaved with subtilisin

[17,20,30]. In agreement with these data, in the present

study, ECP-cleaved Mg-G-actin polymerized more

Fig. 2. Temperature dependences of the excess heat capacity (Cp)

of intact (curve 1), ECP-cleaved (curve 2) and subtilisin-cleaved

(curve 3) ATP-Ca-G-actins. The actin concentration was 24 lM.

Other conditions: 2 mM Hepes (pH 7.6), 0.2 mM CaCl2 and 0.2 mM

ATP. The inset shows representative SDS ⁄ PAGE patterns of intact

(lanes 1 and 1¢), ECP-cleaved (lanes 2 and 2¢) and subtilisin-cleaved

(lanes 3 and 3¢) G-actin before (lanes 1, 2 and 3) and after heating

in the calorimetric cell up to 80 �C (lanes 1¢, 2¢ and 3¢). Note that

the positions of actin (lanes 1 and 1¢) and its C-terminal fragments

produced by ECP (36 kDa) (lanes 2, 2¢) or by subtilisin (35 kDa)

(lanes 3 and 3¢) remain unchanged after the heating–cooling

procedure.

Table 1. Calorimetric parameters obtained from the DSC data for

intact, ECP-cleaved and subtilisin-cleaved G-actins. The parameters

were extracted from Figs 2 and 3. The error of the given values of

Tm did not exceed ±0.2 �C. The relative error of the given values of

DHcal did not exceed ±10%.

G-actin Nucleotide Cation Tm (�C) DHcal (kJÆmol)1)

Intact ATP Ca2+ 61.2 585

Intact ATP Mg2+ 59.9 570

Intact ADP Mg2+ 48.8 340

ECP-cleaved ATP Ca2+ 55.9 370

ECP-cleaved ATP Mg2+ 53.6 390

ECP-cleaved ADP Mg2+ 44.5 145

Subtilisin-cleaved ATP Ca2+ 57.0 335

Thermal unfolding of cleaved actin A. V. Pivovarova et al.


slowly than subtilisin-cleaved Mg-G-actin, which, in

turn, demonstrated slower polymerization than intact,

noncleaved Mg-G-actin. Nevertheless, light-scattering

measurements showed complete polymerization of all

the Mg-G-actin species to Mg-F-actin after 1.5 h of

incubation with 100 mm KCl and 1 mm MgCl2 in the

presence of 1 mm ATP (Fig. 4, inset).

Figure 4 shows that Mg-F-actin obtained from the

cleaved Mg-G-actin is much less thermostable than

noncleaved Mg-F-actin, and a decrease in the thermal

stability is even more pronounced than in the case of

G-actin. The thermal transitions of ECP-cleaved and

subtilisin-cleaved F-actin are shifted to a lower temper-

ature, by 11.3 and 8.8 �C, respectively, compared to

that of intact F-actin (Table 2). Importantly, a pro-

nounced difference is observed between the thermal

transitions of ECP-cleaved and subtilisin-cleaved

F-actin (Fig. 4). ECP-cleaved F-actin unfolds not only

at lower temperature (58.6 versus 61.1 �C), but also

with a much lower cooperativity. The width at the

half-height of the thermal transition, which can serve

as a relative measure for cooperativity of the transi-

tion, was equal to 8.5 �C for ECP-cleaved Mg-F-actin

and 4.3 �C for subtilisin-cleaved Mg-F-actin. Thus,

Fig. 4. DSC curves of Mg-F-actin assembled from intact (curve 1),

ECP-cleaved (curve 2) and subtilisin-cleaved (curve 3) ATP-Mg-G-

actin. The actin concentration was 24 lM. Other conditions: 20 mM

Hepes (pH 7.3), 0.1 M KCl, 1 mM MgCl2 and 0.7 mM ADP. The

inset shows time courses of polymerization of intact (curve 1),

ECP-cleaved (curve 2) and subtilisin-cleaved (curve 3) actins. Poly-

merization was monitored by recording light-scattering intensity at

350 nm upon the addition of 0.1 M KCl and 1 mM MgCl2 to ATP-

Mg-G-actins.

Fig. 3. DSC curves of intact G-actin (A) and ECP-cleaved G-actin

(B) with different tightly bound nucleotide and cation: ATP-Ca-G-

actin, ATP-Mg-G-actin and ADP-Mg-G-actin. The actin concentration

was 24 lM. Other conditions: 2 mM Hepes (pH 7.6), 0.2 mM CaCl2

or MgCl2, and 0.2 mM ATP or ADP.

Table 2. Calorimetric parameters obtained from the DSC data for

Mg-F-actin assembled from intact, ECP-cleaved and subtilisin-

cleaved Mg-G-actin. The parameters were extracted from Figs 4

and 5. The error of the given values of Tm did not exceed ± 0.2 �C.

The relative error of the given values of DHcal did not exceed

±10%.

Mg-F-actin Stabilizer Tm (�C) DHcal (kJÆmol)1)

Intact – 69.9 650

Intact Phalloidin 82.5 1065

Intact AlF4� 83.4 800

Intact Phalloidin + AlF4� 90.8 1080

ECP-cleaved – 58.6 525

ECP-cleaved Phalloidin 68.5 655

ECP-cleaved AlF4� 70.3 415

ECP-cleaved Phalloidin + AlF4� 81.7 690

Subtilisin-cleaved – 61.1 415

Subtilisin-cleaved Phalloidin 76.5 635

Subtilisin-cleaved AlF4� 76.4 720

Subtilisin-cleaved Phalloidin + AlF4� 84.4 780



although both cleaved G-actins unfold similarly

(Fig. 2), a pronounced difference in the thermal

unfolding between ECP-cleaved and subtilisin-cleaved

actin is revealed when these actins are polymerized into

filaments.

Stabilization of the cleaved F-actin by phalloidin

and aluminum fluoride

It is well known that cyclic heptapeptide phalloidin

binds to F-actin with very high affinity at the interface

of three adjacent actin protomers [31,32] and stabilizes

actin filaments (i.e. it significantly increases the thermal

stability of F-actin) [25,28,33–35]. A very similar stabi-

lizing effect was observed in the presence of Pi analogs,

aluminum fluoride (AlF�4 ) or beryllium fluoride (BeFx)

[25,28,34,36], which form complexes with F-actin

subunits that mimic their ADP-Pi state. The stabilizing

effects of phalloidin and AlF4� (or BeFx) were similar

but independent of each other because simultaneous

addition of both stabilizers caused an additional

increase in the thermal stability of F-actin [28,34].

The subsequent experiments were designed to inves-

tigate the effects of the two F-actin stabilizers, phalloi-

din and AlF4� , on the thermal unfolding of F-actin

specifically cleaved within the D-loop. In agreement

with previous studies [25,28,29,34], the binding of

phalloidin or AlF4� significantly increased the thermal

stability of Mg-F-actin. Both stabilizers shifted the

maximum of the F-actin thermal transition from

69.9 �C to 82–83 �C (Table 2), and their simultaneous

addition increased the Tm up to � 91 �C (Fig. 5A).

Similar to intact F-actin, both cleaved F-actin species

are significantly stabilized by phalloidin and AlF4�

(Fig. 5B,C). Each of these stabilizers increased the Tm

of the cleaved F-actin, by 10–12 �C for ECP-cleaved

F-actin and by � 15 �C for subtilisin-cleaved F-actin

(Table 2), and their simultaneous addition resulted in

an additive effect that is expressed in the further

increase of the Tm value by � 12–13 �C (Fig. 5B) or

8 �C (Fig. 5C). However, in all these stabilized states,

the Tm value for the cleaved F-actin was significantly

lower than that of intact F-actin, by 9–14 �C for ECP-

cleaved F-actin and by 6–7 �C for subtilisin-cleaved

F-actin (Table 2). This means that ECP-cleaved F-actin

is less thermostable than subtilisin-cleaved F-actin not

only in the absence of stabilizers (Fig. 4), but also in

the presence of phalloidin and AlF4� (Fig. 5B,C).

There are also other distinct differences between

ECP-cleaved F-actin and subtilisin-cleaved F-actin,

whose thermal denaturation is more similar to that of

intact F-actin. First, along with the main transition at

68.5 �C, the DSC profile of the phalloidin-stabilized

ECP-cleaved F-actin demonstrated a pronounced

shoulder at � 60 �C (Fig. 5B). Second, in the presence

of AlF4� , this cleaved F-actin demonstrated, along with

the main thermal transition at 70 �C, a clear peak at

� 57 �C corresponding to the thermal unfolding of this

protein in the absence of AlF4� (Fig. 5B). This suggests

a much lower affinity of ECP-cleaved F-actin for

phalloidin and AlF4� than that in intact and subtilisin-

cleaved F-actin. To test this assumption, we investi-

gated the thermal unfolding of ECP-cleaved F-actin in

the presence of different concentrations of phalloidin

and AlF4� (Fig. 6).

At relatively low phalloidin ⁄ actin molar ratio of

1 : 4, two peaks are observed on the DSC profile

Fig. 5. DSC curves of intact Mg-F-actin (A), ECP-cleaved Mg-F-

actin (B) and subtilisin-cleaved Mg-F-actin (C) stabilized by phalloidin

or AlF4� , or simultaneously by both stabilizers. Concentrations of

stabilizers: 24 lM phalloidin and 1 mM AlF4� (5 mM NaF and 1 mM

AlCl3). Other conditions were as described in Fig. 4.



(Fig. 6A), and the large peak with Tm at 58.6 �C corre-

sponds to the nonstabilized ECP-cleaved F-actin (i.e. it

reflects the thermal unfolding of those actin protomers,

which are not affected by phalloidin). This means that

effect of phalloidin on the thermal stability of ECP-

cleaved F-actin is much less cooperative than in the

case of intact F-actin, when one bound phalloidin was

shown to stabilize up to seven neighboring protomers

in the actin filament [37]. The peak of nonstabilized

actin disappeared with an increase in the phalloi-

din ⁄ actin molar ratio (Figs 5B and 6A). However, the

pronounced shoulder at � 61–65 �C was observed on

the DSC profile of ECP-cleaved F-actin even in the

presence of a three-fold molar excess of phalloidin

(Fig. 6A), thus suggesting that protomers of phalloi-

din-stabilized F-actin exist in two structural states with

different thermal stability.

At a low concentration of AlF4� (0.1 mm), we again

observed a pronounced peak at 58.6 �C corresponding

to the nonstabilized ECP-cleaved F-actin (Fig. 6B).

Thus, a much higher concentration of AlF4� (more

than 1 mm) is required to achieve complete thermal

stabilization of ECP-cleaved F-actin compared to

intact F-actin, for which full stabilization was observed

even in the presence of 50 lm AlF4� [36]. This reflects

at least an order of magnitude lower affinity of the

cleaved F-actin to AlF4� .

Importantly, upon simultaneous addition of AlF4�

and phalloidin, we observed neither the peak of

nonstabilized actin protomers, nor the shoulder char-

acteristic of phalloidin-stabilized ECP-cleaved F-actin

(Fig. 6B). These results suggest that the binding of

AlF4� to ADP-F-actin substantially modifies the struc-

tural state of cleaved actin subunits stabilized by phal-

loidin or phalloidin increases the affinity of the cleaved

actin subunits to AlF4� .

Discussion

The data reported in the present study show that

cleavage of actin between Gly42-Val43 or Met47-Gly48

within the D-loop strongly decreases the thermal

stability both of monomers and polymers. According to

previous studies, these cleavages increased the rate of

the nucleotide exchange on the cleaved G-actin and its

susceptibility to limited proteolysis, probably as a result

of the transition of the nucleotide-binding cleft to a

more open conformation [5,17,22]. The relationship

between the conformation of the D-loop and the nucleo-

tide-binding cleft was recently demonstrated in metady-

namic simulations experiments [13]. We assume

therefore that the decrease in the thermal stability

observed by DSC on actin species cleaved within the

D-loop is associated with opening of the cleft.

Does the thermal stability of G-actin reflect the

conformational state of the nucleotide-binding

cleft?

An intact actin structure is maintained by the presence

of high-affinity cation and nucleotide tightly bound in

the interdomain cleft; removal of the nucleotide or cat-

ion results in actin denaturation. Therefore, the stabil-

ity of actin depends on the affinity of the tightly

bound cation and nucleotide that involves both pro-

tein–ligand interaction and conformation of the inter-

domain cleft. Upon heating, irreversible unfolding of

G-actin is preceded by reversible loss of the nucleo-

tide–cation complex [23]. Obviously, the more tightly

nucleotide and cation are bound in the interdomain

cleft and the more ‘closed’ is the cleft, the higher the

temperature needed to remove them from the cleft and

to induce thermal unfolding of G-actin. The relative

Fig. 6. DSC curves for ECP-cleaved Mg-F-actin (24 lM) either in

the presence of phalloidin (Ph) at different concentrations (6, 12 or

72 lM) (A), or in the presence of 0.1 mM AlF4� in the absence or in

the presence of 24 lM Ph, and in the presence of 0.5 mM

AlF4� + 24 lM Ph (B). Other conditions were as described in Fig. 4.



affinity of G-actin for ATP is much higher than for

ADP [4], and the interdomain cleft is suggested to be

in a more open conformation in the ADP-bound state

than in the ATP-bound state [5,12,13]. In agreement

with this and with previous studies [23,24], the results

obtained in the present study show that ADP-G-actin

is much less thermostable than ATP-G-actin (Fig. 3A

and Table 1). ATP-G-actin is less thermostable with

bound Mg2+ than with Ca2+ [23] (Table 1), and this

reduction in stability may be explained by the lower

affinity of ATP-G-actin for Mg2+ than for Ca2+

[4,38]. Thus, the ligand-dependent thermal stability of

actin monomer can be accounted for by the different

affinity of these ligands to actin. However, the thermal

stability of the cleaved actins is lower than the corre-

sponding stability of non-modified actin both in the

ATP- and ADP-states. This cannot be explained by

the different affinity but suggests that the thermal sta-

bility of G-actin may depend on the conformation of

the nucleotide-binding cleft. This suggestion is sup-

ported by the studies on the effects of actin-binding

proteins on actin structure.

Actin-binding proteins profilin and cofilin, when

bound to G-actin between subdomains 1 and 3, have

antagonistic effects on the conformation of the nucleo-

tide-binding cleft. Profilin stabilizes the ‘open’ confor-

mation of the cleft [7,39,40], whereas cofilin appears to

lock the cleft in its ‘closed’ conformation [39–42]. Pre-

vious studies on the thermal unfolding of G-actin

showed that profilin binding decreased the actin ther-

mal stability [23], whereas significant stabilization of

G-actin was observed in its complexes with cofilin

[25,26]. Stabilization of G-actin was also observed in

the complexes of G-actin with thymosin b4 [27] and

gelsolin segment 1 [24], which appear to induce confor-

mational transitions closing the nucleotide-binding

cleft [6,27,43,44].

Thus, the increased thermal stability of G-actin

appears to correspond to the closed conformation of

the nucleotide-binding cleft, whereas the decreased

thermal stability is a feature of the actin with the open

cleft conformation. The cleavage within the D-loop

enhances the nucleotide exchange [17] and increases

accessibility of the cleft to limited proteolysis [5], which

characterizes the cleft opening. It is therefore likely

that the decreased thermal stability of G-actin cleaved

within the D-loop also results from the opening of the

nucleotide-binding cleft in these actin species.

It is noteworthy that the replacement of tightly

bound ATP by ADP in ECP-cleaved G-actin induces

an additional decrease in the thermal stability of this

actin species already destabilized by the cleavage

within the D-loop (Fig. 3B and Table 1). This suggests

that the nucleotide-binding cleft is highly dynamic and

cannot be simply open or closed but rather should be

considered as being more open or more closed. In

these terms, by analogy with the ‘superclosed’ state

recently revealed in ATP-G-actin by molecular dynam-

ics simulations [12], the nucleotide-binding cleft of

ADP-G-actin cleaved within the D-loop appears to

adopt the extra open conformation.

Comparison of the effects produced by the

cleavage of the D-loop with ECP and subtilisin

Although the cleavages of the D-loop between Gly42-

Val43 and Met47-Gly48 decreased the thermal stability

of G-actin to a similar extent (Fig. 2), the effects of

the cleavages became quite different when the cleaved

actins were polymerized into filaments. The thermal

stability of F-actin assembled from ECP-cleaved actin

was noticeably less than that of subtilisin-cleaved

F-actin (Fig. 4). These results are consistent with the

earlier observed effects of these cleavages on the sus-

ceptibility of the nucleotide-binding cleft to limited

proteolysis with trypsin [5,17]. In the cleaved G-actins,

susceptibility of trypsin cleavage sites at Arg62 and

Lys68 in the nucleotide-binding cleft was increased

similarly [17]. After polymerization, these sites became

almost inaccessible for trypsin in intact F-actin and

only slightly accessible for trypsin in subtilisin-cleaved

F-actin. By contrast, F-actin assembled from ECP-

cleaved G-actin was easily fragmented by trypsin.

These observations indicate that the open conforma-

tion of ECP-cleaved actin was preserved upon poly-

merization, whereas F-actin assembled from subtilisin-

cleaved monomers more closely resembled intact

F-actin than ECP-cleaved F-actin [17]. Thus, the lower

thermal stability of ECP-cleaved versus subtilisin-

cleaved F-actin corresponds to a more open nucleo-

tide-binding cleft.

According to the recent model of actin filament [45],

the N-terminal part of the D-loop is located at the

inter-monomer interface, participating both in the

intra-strand contacts between actin subunits along

the filament and in the lateral contacts stabilizing the

inter-strand interaction, whereas the C-terminal part of

the loop is not involved in the inter-strand contacts.

Recently, this structural difference was supported in

mutational cross-linking experiments showing that the

N-terminal part of the D-loop (residues 41–45) is in

close proximity to residue 265 of the actin subunit in

the opposite strand and can be easily cross-linked to

this residue, whereas the rate and extent of the cross-

linking reaction strongly declined for the C-terminal

residues of the D-loop [46]. Therefore, the inter-strand



contacts of the N-terminal part of the D-loop appear

to play a crucial role in stabilization of the actin fila-

ment [17,30,47]. The cleavage of the D-loop between

Gly42 and Val43 impairs these contacts [47], and this

may explain why the cleavage of the D-loop in its

N-terminal part with ECP more strongly destabilizes

F-actin than cleavage by subtilisin between Met47 and

Gly48 in the C-terminal part of the loop. It is also

important that the cleavage with ECP did not affect

the filament length but more strongly enhanced the

turnover rate of polymer subunits than the cleavage

with subtilisin [17,30]. Thus, the low thermal stability

of F-actin assembled from ECP-cleaved monomers

strongly correlates with the high dynamics of this actin

species [17], supporting the idea of the monomer disso-

ciation being the first step of thermal inactivation of

F-actin [28,29].

Although both cleaved actins are stabilized with

phalloidin and AlF4� , stabilization of ECP-cleaved

F-actin demonstrates specific features that are not

characteristic of subtilisin-cleaved or intact actin. The

most interesting features are the extremely low affinity

of ECP-cleaved F-actin to AlF4� and the pronounced

shoulder observed on the DSC profile of this actin spe-

cies even in the presence of a three-fold molar excess

of phalloidin (Figs 5B and 6). This suggests that pro-

tomers of phalloidin-stabilized cleaved F-actin exist in

two different structural states. Phalloidin binds to

F-actin at the interface of three adjacent actin protom-

ers [31] and appears to stabilize actin filament in two

inter-related ways: by stabilizing lateral interactions

between the two filament strands and by inducing con-

formational changes in actin subunits resulting in the

state of the nucleotide-binding cleft being similar to

that in ATP-actin filaments without phalloidin [32]. It

is plausible that the shoulder on the DSC profile of

phalloidin-stabilized ECP-cleaved F-actin belongs to a

population of the protomers in which the conforma-

tional effect of phalloidin is not completed. This expla-

nation, although requiring further examination with

independent approaches, is supported by the disap-

pearance of the shoulder after the addition of AlF4�

(Fig. 6B). This Pi analog (as well as another analog,

BeFx) is known to bind to Pi site in the nucleotide-

binding cleft and mimic ADP-Pi or ATP actin fila-

ments [48], thus stabilizing the filament by closing the

cleft in actin subunits [49,50]. Hence, the increase in

the thermal stability of ECP-cleaved F-actin and the

disappearance of the shoulder on the DSC profile can

be accounted for by the combined effect of phalloidin

and AlF4� on the nucleotide-containing cleft. Accord-

ingly, the phalloidin-induced effect may increase the

affinity of AlF4� to actin, whereas AlF4� -induced clo-

sure of the cleft diminishes the population of the su-

bunits remaining nonstabilized by phalloidin via its

effect on the cleft conformation. This interpretation is

consistent with recently published DSC data showing

that cooperative effect of phalloidin on the thermal

stability of F-actin becomes noncooperative in the

presence of AlF4� [51].

Phalloidin can stabilize F-actin with a very high coo-

perativity, with the half-maximal effect being observed

at a phalloidin ⁄actin molar ratio of 1 : 20 [52]. In the

DSC experiments on intact actin [37], only 10–15% of

actin protomers remained unaffected by phalloidin at a

phalloidin ⁄ actin molar ratio of 1 : 4. By contrast, more

than half of subunits of ECP-cleaved F-actin remained

nonstabilized by phalloidin under the same conditions

(Fig. 6A), consistent with a reduced cooperativity in

the effect of phalloidin on the steady-state ATPase

activity of ECP-cleaved actin [17]. Taken together with

the evidence concerning the critical role of the lateral

contacts for stabilization of filaments assembled from

ECP-actin monomers [47], these data allow us to

assume that only the effect of phalloidin on the con-

formation of the nucleotide-binding cleft is coopera-

tive; it is propagated along the filament by allosteric

interactions between phalloidin-bound and free pro-

tomers. By contrast, the stabilizing effect of phalloidin

on the lateral inter-strand interactions is noncoopera-

tive; it requires direct binding of phalloidin to actin

protomers.

According to this interpretation, an explanation for

the appearance of the pronounced shoulder on the

DSC profile of the ECP-cleaved F-actin stabilized by

phalloidin (Fig. 6A) can be proposed. This shoulder

appears to reflect the thermal unfolding of the actin

protomers whose cleft remains open, and therefore

they are stabilized only by lateral inter-strand interac-

tions induced by the direct binding of phalloidin. On

the other hand, the main transition at 68.5 �C(Fig. 6A) most likely corresponds to the thermal

unfolding of actin subunits that are stabilized not only

by the inter-strand interactions, but also by phalloidin-

induced closing of the nucleotide-binding cleft.

In conclusion, the results obtained in the present

study suggest that the thermal stability of actin, regard-

less of whether it is modified by limited proteolysis or

by stabilizers, depends on the conformation of the

interdomain nucleotide-binding cleft. Accordingly, the

lower thermal stability of subtilisin- or ECP-cleaved

actin compared to intact actin supports the idea [5,13]

and also provides additional experimental evidence for

a distinct allosteric relationship between conformation

of the D-loop and the state of the nucleotide-binding

cleft.



Experimental procedures

Reagents

Subtilisin (type VIII bacterial protease), ATP, ADP,

EGTA, Hepes, phenylmethylsulfonyl fluoride, KCl, CaCl2,

MgCl2, AlCl3, NaF and phalloidin were purchased from

Sigma Chemical Co. (St Louis, MO, USA); hexokinase was

kindly provided by Dr N. Yu. Goncharova (Department of

Biochemistry, School of Biology, Moscow State University,

Russia).

Protein preparations

Rabbit skeletal muscle actin was prepared from acetone-

dried muscle powder according to the method of Spudich

and Watt [53]. G-actin was stored in buffer containing

2 mm Tris-HCl (pH 8.0), 0.2 mm ATP, 0.2 mm CaCl2,

0.5 mm b-mercaptoethanol and 0.03% NaN3 (buffer G).

The actin molar concentration was determined by monitor-

ing A290 using an E1% of 6.3 cm)1 [54] and a molecular

mass of 42.3 kDa. ECP-cleaved G-actin was obtained as

described previously [17,30]. Ca-G-actin (3.0 mgÆmL)1) was

digested at an enzyme ⁄ protein mass ratio of � 1 : 100 for

4 h at 25 �C and then overnight at 4 �C. Because actin

cleaved with ECP between Gly42 and Val43 is fairly resis-

tant to further proteolysis by this protease, it was not nec-

essary to use any protease inhibitor. The cleaved actin was

used within 8–10 h. Subtilisin-cleaved actin was prepared

essentially as described by Schwyter et al. [18]. Ca-G-actin

(3 mgÆmL)1) was digested for 1 h at an enzyme ⁄ proteinmass ratio of 1 : 500 at 25 �C, and the proteolysis was

stopped with 2 mm phenylmethylsulfonyl fluoride. The

cleaved actin preparations were analyzed by SDS ⁄PAGE

[55]. Usually, more than 85% of actin was cleaved. It is

important that the main part of the noncleaved actin

appears to correspond to small aggregates of unfolded (so-

called ‘inactivated’) G-actin [56], in which the D-loop

becomes almost inaccessible to proteolytic cleavage [57].

ATP-Ca-G-actin was transformed into ATP-Mg-G-actin

by a 3–5 min of incubation with 0.2 mm EGTA ⁄ 0.1 mm

MgCl2 at 25 �C. To obtain ADP-Mg-G-actin, the actin-

bound ATP was converted into ADP by incubation of

ATP-Mg-G-actin with 0.8 mm ADP, 1 mm glucose and

hexokinase (8 UÆmL)1) for 2 h at 4 �C [5]. It is known that,

under similar conditions, only � 0.4% of ATP was deter-

mined in the actin samples after 1 h of incubation with glu-

cose and hexokinase [58].

Intact, ECP-cleaved and subtilisin-cleaved Mg-G-actins

(3 mgÆmL)1) were polymerized by the addition of 100 mm

KCl and 1 mm MgCl2 in the presence of 1 mm ATP. Poly-

merization was monitored by an increase in intensity of

light scattering at 90� measured at 350 nm on a Cary

Eclipse fluorescence spectrophotometer (Varian Australia

Pty Ltd, Mulgrave, Victoria, Australia).

Stabilization of F-actin (24 lm) by phalloidin or by alu-

minum fluoride (AlF4� ) was performed as described previ-

ously [25,28], by the addition of 6–72 lm phalloidin or 0.1–

1.0 mm AlCl3 in the presence of 5 mm NaF and 0.7 mm

ADP.

DSC

DSC experiments were performed on a DASM-4M differen-

tial scanning microcalorimeter (Institute for Biological

Instrumentation, Pushchino, Russia) as described previously

[25,28,29,36]. All measurements were carried out at a scan-

ning rate of 1 KÆmin)1. The experiments with G-actin were

performed in 2 mm Hepes, pH 7.6, containing 0.2 mm CaCl2or MgCl2 and 0.2 mm ATP (or 0.2 mm ADP in the case of

ADP-Mg-G-actin), whereas the thermal unfolding of F-actin

was studied in 20 mm Hepes (pH 7.3), 0.1 m KCl, 1 mm

MgCl2 and 0.7 mm ADP. The final concentration of actin

was 24 lm. The reversibility of the thermal transitions was

assessed by reheating of the sample immediately after cool-

ing from the previous scan. The thermal denaturation of all

actin samples was fully irreversible. Calorimetric traces were

corrected for instrumental background and possible aggre-

gation artifacts by subtracting the scans obtained from the

reheating of the samples. The temperature dependence of

the excess heat capacity was further analyzed and plotted

using Origin software (MicroCal, Northampton, MA,

USA). The thermal stability of actin was described by the

Tm, and DHcal was calculated as the area under the excess

heat capacity function. DSC experiments with different actin

species were performed at least twice with very good repro-

ducibility, and the representative curves are shown.

Acknowledgements

We are grateful to Dr Alevtina Morozova for provid-

ing us with protease ECP32 ⁄ grimelysin. This work was

supported by the Russian Foundation for Basic

Research (grants 09-04-00266 to D.I.L. and 08-04-

00408 to S.Yu.Kh), the Program ‘Molecular and Cell

Biology’ of the Russian Academy of Sciences, and by

the grant from the President of Russian Federation

(grant MK 2965.2009.4 to A.V.P.).

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Specific cleavage of the DNase-I binding loop dramatically decreases the thermal stability of actin