Báo cáo khoa học: A structural basis for the pH-dependence of cofilin F-actin interactions potx

A structural basis for the pH-dependence of cofilin
F-actin interactions
Laurence Blondin
1
, Vasilia Sapountzi
2
, Sutherland K. Maciver
1
, Emeline Lagarrigue
2
, Yves Benyamin
1
and Claude Roustan
1
1
Laboratoire de motilite
´
cellulaire, Universite
´
de Montpellier, France;
2
Genes and Development Group, Department of Biomedical
Sciences, University of Edinburgh, Scotland
A marked pH-dependent interaction with F-actin is an
important property of typical members of the actin
depolymerizing factor (ADF)/cofilin family of abundant
actin-binding proteins. ADF/cofilins tend to bind to
F-actin with a ratio of 1 : 1 at pH values around 6.5, and
to G-actin at pH 8.0. We have investigated the mechan-
ism for the pH-sensitivity. We found no evidence for
pH-dependent changes in the structure of cofilin itself, nor

for the interaction of cofilin with G-actin. None of the
actin-derived, cofilin-binding peptides that we had previ-
ously identified [Renoult, C., Ternent, D., Maciver, S.K.,
Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C.
(1999) J. Biol. Chem. 274, 28893–28899] bound cofilin in a
pH-sensitive manner. However, we have detected a
conformational change in region 75–105 in the actin
subdomain 1 by the use of a peptide-directed antibody. A
pH-dependent conformational change has also been
detected spectroscopically in a similar peptide (84–103) on
binding to cofilin. These results are consistent with a
model in which pH-dependent motion of subdomain 1
relative to subdomain 2 (through region 75–105) of actin
reveals a second cofilin binding site on actin (centered
around region 112–125) that allows ADF/cofilin associ-
ation with the actin filament. This motion requires salt in
addition to low pH.
Keywords: cofilin; actin; pH dependency; synthetic peptide;
actin antibodies.
The ADF/cofilins are a family of actin-binding proteins that
are pivotally involved in both the polymerization and
depolymerization of actin filaments, most notably in the
advancing lamellae of motile cells [1,2]. Cell motility,
through the actin-based cytoskeleton, is tightly controlled
by the interplay of a variety of signaling pathways. The
importance of the contribution of ADF/cofilins to cell
motility is reflected in their being regulated by many of these
signals, including phosphorylation [3], polyphosphoinosi-
tides [4–6], the presence of other actin-binding proteins [7–
10] and pH [11–13]. Evidence for the regulation of the ADF/

cofilins by pH has been present both in vitro [11–14] and in
living cells [15]. Most members of the ADF/cofilin family
show a complex pH-dependent behaviour with respect to
F-actin binding; exceptions are depactin from sea urchin
eggs [16] and actophorin [17] from the soil amoeba
Acanthamoeba. ADF/cofilins in general tend to bind to
F-actin around pH 6.5 and to G-actin around pH 8.0
[6,11,18], but actophorin binds rabbit skeletal muscle
F-actin at both pH extremes [17]. Actin solutions can be
reversibly transformed from the G to F state by changes in
pH in the presence of cofilin [6,11,19]. The F-actin bound by
cofilin at low pH has several properties distinct from that of
F-actin alone. These cofilin–actin filaments are short [19],
have an increased helical twist [20] and do not bind
phalloidin [8,12], caldesmon [8] or tropomyosin [7,10,21].
The study of the pH sensitivity of the actin–cofilin
interaction is complicated by the fact that actin itself is
pH-sensitive across the same range. The spontaneous
polymerization of actin is more rapid at pH 6.5 than at
pH 8.0 [22] and there appears to be a difference in
conformation of G-actin at the two pH extremes [23].
Transients in intracellular pH occur in a variety of
situations such as chemotaxis [24], mitosis, depolarization
[25] and ischemia [26]. The actin–cofilins are typically
concentrated at the leading edge of cells [5,27,28] and the cell
cortex, regions that are especially likely to experience local
fluctuations in pH [25]. The lammelae of alkalized macro-
phages ÔhyperruffleÕ, whereas ruffling ceased on intracellular
acidification [29], as expected from the properties that the
ADF/cofilins display in vitro.

The position and geometry with which ADF/cofilins bind
F-actin has been controversial. Image reconstructions have
placed cofilin on the surface of the filament, between
subdomain 1 of one actin monomer and subdomain 2 of
the longitudinally associated monomer, immediately toward
the barbed end of the filament [20,30,31]. Our previous
studies [32,33] argue that cofilin is not on the surface of the
filament but is instead buried between two longitudinally
associated monomers within the filament, and that subdo-
main 2 from one monomer and subdomain 1 from the other
are pushed apart. This results in the increased twist of the
Correspondence to C. Roustan, UMR 5539[CNRS] UM2 CC107,
Universite
´
de Montpellier 2, Place E. Bataillon CC107,
34095 Montpellier Cedex 5, France.
Fax: + 33 0467144927, E-mail:
Abbreviations: ADF, actin depolymerizing factor; FITC, fluorescein
5-isothiocyanate; RITC, rhodamine isothiocyanate; 1,5-I-AEDANS,
N,-iodoacetyl-N¢-[sulfo-1-naphthyl]-ethylenediamine; G-actin,
monomeric actin; F-actin, filamentous actin.
Note: web pages are available at />umr5539/, , .
ac.uk/research/smaciver/index.htm
(Received 9 May 2002, accepted 27 June 2002)
Eur. J. Biochem. 269, 4194–4201 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03101.x
actin filament observed first by McGough and coworkers
[20] and subsequently by others [31], and the thrusting
forward of subdomain 2 with respect to the rest of the
monomer.
In this report we study the pH-dependence of the actin–

cofilin interface and provide evidence for a pH-dependent
movement of subdomain 1 that may be involved in the pH-
dependence of the interaction of cofilin with actin.
EXPERIMENTAL PROCEDURES
Proteins and peptides
Rabbit skeletal muscle actin was isolated from acetone
powder [34]. Human cofilin was produced in E. coli
[BL21(DE3)], transfected with a T7-based vector, pMW172,
carrying a human nonmuscle cofilin encoding cDNA
fragment and purified as described previously [13,35]. Cofilin
labeling with fluorescein isothiocyanate (FITC) was carried
out by incubating the reagent (dissolved in N,N-dimethyl-
formamide) with the protein in a molar ratio of 1 : 4. The
coupling reaction was carried out in 50 m
M
NaHCO
3
buffer, pH 8.5, for 3 h, and excess reagent removed by gel
filtration (PD-10, Amersham Pharmacia Biotech.) and
equilibrated with the same buffer. The stoichiometry of
the labeling was determined to be 0.7 mol FITC per mol
cofilin. The procedure for Rhodamine isothiocyanate
(RITC) labeling of cofilin or actin was similar, except that
thereactionwasperformedusinga3molarexcessof
reagent. Antibodies directed towards cofilin or 75–105
peptide coupled to hemocyanin were elicited in rabbits [36].
They were labeled with Oregon green (Molecular Probes) by
thesameproceduredescribedforFITCexceptthata20
molar excess of reagent was used. IgGs labeled with alkaline
phosphatase were purchased from Sigma.

Synthetic peptides derived from actin sequences were
prepared on solid phase support using a 9050 Milligen
PepSynthesizer (Millipore, U.K.) according to the Fmoc/tBu
system. The crude peptides were deprotected and thoroughly
purified by preparative reverse-phase HPLC. The purified
peptides were shown to be homogenous by analytical HPLC.
Electrospray mass spectra, carried out in the positive ion
mode using a Trio 2000 VG Biotech Mass spectrometer
(Altrincham, UK), were in line with the expected structures.
Peptides were labeled at the cysteine residue with N-iodo-
acetyl-N¢-[sulfo-1-naphthyl]-ethylenediamine (1.5-I-AE-
DANS) or at amino groups by FITC [37,38]. Excess
reagent was eliminated by sieving through a Biogel P2 col-
umn equilibrated with 0.05
M
NH
4
HCO
3
buffer, pH 8.0.
Immunological techniques
ELISA [39], previously described in detail [40], was used to
monitor the interaction between coated peptides and cofilin.
Peptides (5 lgÆmL
)1
)in50m
M
NaHCO
3
/Na

2
CO
3
,pH9.5,
were immobilized on plastic microtiter wells. The plate was
then saturated with 0.5% gelatin and 3% gelatin hydroly-
sate, in 140 m
M
NaCl, 50 m
M
Tris buffer, pH 7.5. Binding
was monitored at 405 nm using alkaline phosphatase-
labeled IgGs (dilution 1 : 1000). Control assays were carried
out in wells saturated with the mixture of gelatin and gelatin
hydrolysate used alone. Each assay was conducted in
triplicate and the mean value plotted after subtraction of
nonspecific absorption. The binding parameters (apparent
dissociation constant K
d
and the maximal binding A
max
)
were determined by non linear fitting A ¼ A
max
· [L]/
(K
d
+ [L]) where A is the absorbance at 405 nm and L the
ligand concentration, by using the
CURVE FIT

software
developed by Kevin Raner software, Victoria, Australia.
Additional details on the different experimental conditions
are given in the figure legends.
Fluorescence measurements
Fluorescence experiments were conducted using a LS 50
Perkin-Elmer luminescence spectrometer. Spectra for FITC,
Oregon green or RITC were obtained with the excitation
wavelength set at 470, 480 and 540 nm, respectively.
Fluorescence changes were deduced from the area of the
emission spectra of FITC or Oregon green between 510 and
530 and 570–590 nm for RITC. The parameters K
d
(apparent dissociation constant) and A
max
(maximum
effect) were calculated by nonlinear fitting of the experi-
mental data points.
Actin binding to immobilized cofilin
Recombinant human nonmuscle cofilin was coupled to
cyanogen-activated Sepharose 4B beads (Amersham Phar-
macia Biotech.) according to the manufacturer’s recom-
mendations. Excess reactive groups were quenched by
washing with 0.1
M
Tris buffer, pH 8.0. Prior to actin-
binding experiments the beads were washed in Buffer
G–ADP at either pH 6.5 (10 m
M
imidazole, 0.1 m

M
ADP,
0.2 m
M
CaCl
2
,0.2m
M
dithiothreitol and 1 m
M
NaN
3
)or
pH 8.0 (10 m
M
Tris, 0.1 m
M
ADP, 0.2 m
M
CaCl
2
,0.2m
M
dithiothreitol and 1 m
M
NaN
3
). ADP–G-actin was made
from ATP–actin by incubation with hexokinase (Sigma)
and glucose [41] before being added to the indicated total

concentration. The beads were collected by centrifugation
after incubation. The amount of actin bound to the beads
and remaining in the supernatant was measured by scanning
SDS/PAGE gels.
Analytical methods
Protein concentrations were determined by UV absorbency
using a Varian MS 100 (Varian SA, Les Ulis, France), and a
Pharmacia Ultraspec 2000 spectrophotometer. For cofilin
the absorbance was measured at 280 nm, where one
absorbance unit is equivalent to 74 l
M
. For actin solutions,
the absorbance was measured at 290 nm where one
absorbance unit is equivalent to 38 l
M
.SDS/PAGEwas
carried out on 15% gels as described previously [42]
and stained with Coomassie blue R-250.
RESULTS
pH and F-actin
In a previous study [33] we have shown that at pH 6.5,
FITC labeled cofilin binds to G- and F-actin, but a change
in the fluorescence intensity of FITC occurs only when
labeled cofilin interacts with F-actin. In the present study,
similar experiments were performed at two pH values (6.5
and 8.0) for comparison. G-actin and FITC–cofilin were
Ó FEBS 2002 pH-dependence of cofilin–actin interaction (Eur. J. Biochem. 269) 4195
mixed, the addition of salts (0.1
M
KCl and 2 m

M
MgCl
2
)
then induces oligomerization and the fluorescence was
measured at 520 nm as a function of time (Fig. 1). A
significant fluorescent enhancement was observed only at
pH 6.5, immediately after salt addition and before a
significant amount of actin has polymerized [33], even if
the very rapid kinetics of cofilin–actin polymerization at
pH 6.5 are considered [19]. In a control experiment, no
change was observed in the fluorescence intensity of FITC-
labeled cofilin used alone after salt addition to the sample
(data not shown).
Effect of pH on cofilin conformation
The regulation of the cofilin activity by pH occurs in a pH
range suggesting the involvement of histidine residues. In
fact, the single histidine in human and yeast cofilin is not
located in the same position [18,43,44] and more generally
its position is not conserved during evolution. However,
we have looked for a possible structural change in cofilin
induced by pH shift. Two kinds of fluorescence experi-
ments were performed. We have measured the intrinsic
fluorescence of cofilin via its unique tryptophan residue
and the extrinsic fluorescence of RITC covalently linked to
cofilin at various values of pH between 6.5 and 8.0. We
observed no significant changes in fluorescence intensity
(not shown), indicating that the environment of these two
chromophores in cofilin is independent of pH at least in
the range tested.

The pH dependence of the cofilin–G-actin interaction
We then tested for a pH dependence in the interaction of
cofilin with G-actin by two independent methods. G-actin
was labeled with RITC and increasing concentrations of
cofilin were added. In Fig. 2A, we show a decrease in the
fluorescence intensity that is higher at pH 6.5 than pH 8.
Analysis of these data shows that the fluorescence decrease
extrapolated to infinite cofilin concentrations is significantly
different for the two pH (32% and 22% for pH 6.5 and
pH 8.0, respectively, Figs 2A and 3). In contrast, an
apparent K
d
of about 1 l
M
was estimated in both cases.
In a control experiment we observed that the fluorescence of
the RITC-labeled actin is not affected by pH changes within
the same range (not shown). We have confirmed that there
is no difference in affinity between G-actin and cofilin by
measuring the G-actin binding to cofilin immobilizing on
sepharose beads (Fig. 2B). We were able to demonstrate
that this was the case for both ADP and ATP–actin (not
Fig. 1. Cofilin–actin copolymerization. FITC-cofilin (0.5 l
M
)and
G-actin (5 l
M
) were mixed in 50 m
M
Mops, 0.1 m

M
ATP, buffer
pH 6.5 or 8.0, then 0.1
M
KCl, 2 m
M
MgCl
2
were added. FITC-cofilin
fluorescence was monitored at 520 nm versus time at pH 6.5 (—) or
pH 8.0 ( ).
Fig.2. EffectofpHontheinteractionofactinwithcofilin.(A) Effect of pH on the interaction of RITC-actin with cofilin. Binding of RITC-labeled
actin (1.5 l
M
) to cofilin in 50 m
M
Mops, 0.05 m
M
CaCl
2
,0.05m
M
ATP, buffer pH 6.5 or 7.8 was monitored by fluorescence. Changes in the
intensity of the emission spectra were recorded at pH 6.5 (d)and7.8(s), in the presence of increasing cofilin concentrations (between 0 and
3.5 l
M
). An apparent K
d
of about 1 l
M

was estimated in both cases. (B) Binding of ADP-actin to cofilin immobilized on beads in ADP buffer G at
either pH 6.5 (d), or pH 8.0 (s). No significant difference in actin binding was found as pH was varied.
4196 L. Blondin et al.(Eur. J. Biochem. 269) Ó FEBS 2002
shown). In order to deduce a more precise location in the
actin sequence for pH-dependent structural changes
induced by cofilin in F-actin, a peptidic approach was then
carried out.
Actin sequence correlated with pH effect
Two interfaces on the actin surface have been characterized
previously as interacting with cofilin [32,33]. Site 1 includes
the 18–28 sequence and the C-terminal part of the protein,
including the 360–372 sequence. In contrast, site 2 includes
sequences between residues 75–135. These two sites contain
some histidine residues: three residues in the 84–103
fragment and one in the 360–372 fragment. Another
histidine is located in the 38–52 fragment, but this sequence
was previously excluded from the interfaces [33].
The possible effect of pH on the actin site 1 was first
tested using the C-terminal peptides 356–375 or 360–372.
The competition between cofilin towards actin and sequence
360–375 belonging to site 1 was studied at two pH values
(6.6 and 7.5) by ELISA. In this experiment the peptide was
coated to plastic and the binding of cofilin, fixed at 1.8 l
M
,
was monitored in the presence of increasing actin concen-
trations (between 0 and 4.8 l
M
).AsshowninFig.4we
observed a decrease in the cofilin binding in the presence of

actin suggesting that the actin–cofilin complex impedes the
interaction of cofilin with the actin peptide.
The complex formation between the C-terminal sequence
of actin with cofilin was then investigated. Cofilin labeled
with RITC was incubated in the presence of increasing
concentrations of 355–375 actin peptide and the fluores-
cence monitored at pH 6.5 and 8.0. A decrease of fluores-
cence was observed. The binding occurs with similar K
d
of
about 2 l
M
(not shown) and similar fluorescence changes
(34% effect) in both cases (Fig. 3). Then, we have checked
the peptide 355–375 labeled with IAEDANS at its cysteine
residue (at position 374), but the interaction with actin does
not induce any fluorescent change. Therefore, we have
labeled the peptide with IAEDANS corresponding to actin
sequence 360–372 in which an extra cysteine residue was
added at its N-terminal extremity. In the present case, we
observed an increase of the fluorescence intensity upon
cofilin binding. However, the observed variation was similar
for the two pH values used (12% effect) (Fig. 3).
The interaction of the peptide 84–103 corresponding to a
part of site 2 was also checked. As previously reported for
site 1, competition between actin and the peptide 84–103
belonging to site 2 was also investigated. As shown in Fig. 5
we observed a decrease in the binding of cofilin to peptide
84–103 in the presence of increasing actin concentration at
the two pH values tested.

The complex formation between 84 and 103 actin
fragment and cofilin was then determined. To perform
these experiments, either peptide 84–103 was labeled with
Oregon green, or cofilin was labeled with RITC. As shown
in Fig. 6, in both cases and for both pH, the peptide binds to
cofilin with a K
d
of about 3 l
M
. The interaction of cofilin
with Oregon green labeled peptide induces a fluorescence
decrease that is pH-dependant. The maximum effect
extrapolated at infinite cofilin concentration is of 7% at
pH 6.5 and 25% at pH 8.0 (Fig. 3). Similarly, in the second
experiment where fluorescence intensity change of RITC in
labeled cofilin was monitored vs. peptide concentrations, a
decrease of 25% is observed at pH 6.5 and only of 14% at
pH8.0(Figs3and7).
Fig. 3. Effect of pH on the fluorescence changes induced by the inter-
action of cofilin with actin or actin derivative synthetic peptides. Results
are expressed as the maximum fluorescence variation. Enhance-
ment (%) ¼ (A
max
/F
0
) · 100 where A
max
is the maximum fluores-
cence change extrapolated at an infinite ligand concentration, and F
0

the initial fluorescence in the absence of ligand. The experiments were
performedin50m
M
Mops buffer pH 6.5 or pH 8.0 with cofilin in the
presence of different ligands. (A) Oregon green 84–103 pep-
tide + cofilin, (B) rhodamine-labeled cofilin + 84–103 peptide, (C)
rhodamine-labeled G-actin + cofilin, (D) rhodamine-labeled cofi-
lin + 355–375 peptide, (E) Dansylated 360–372 peptide + cofilin.
Fig. 4. Competition binding study between actin fragment 360–372 and
G-actin monitored by ELISA. The binding of cofilin (1.8 l
M
)tocoated
actin peptide of sequence 360–372 in 50 m
M
Mops buffer pH 7.5 (s)
or 6.6 (d), supplemented with 3% gelatin hydrolysate, in the presence
of increasing G-actin concentrations (0–4.8 l
M
). Binding was detected
by using anti-cofilin Ig and monitored at 405 nm.
Ó FEBS 2002 pH-dependence of cofilin–actin interaction (Eur. J. Biochem. 269) 4197
Evidence for a change in the conformation
of actin in the 75–103-actin region
The significance of the modifications observed in the
interface between cofilin and actin upon pH effect was
checked by using a fluorescent probe specific for the site 2 in
actin. We have labeled specific purified antibodies directed
towards 75–105 actin sequence with Oregon green. The
binding of actin to this antibody was monitored at pH 6.5
and 8.0. As shown in Fig. 8, the fluorescence enhancement

is about 4 fold higher at pH 8.0 than pH 6.5 while the
apparent affinities appear unchanged. In contrast, no
change between the two pH, in the fluorescence of
antibodies alone, was obtained. This last result showed that
antibodies interact in a different local environment with the
antigenic epitope located within cofilin site 2 in actin
sequence.
DISCUSSION
The ADF/cofilins are so far unique amongst the many
distinct types of actin-binding proteins in their ability to
alter the twist of actin filaments [20]. This property possibly
explains the extreme cooperativity of F-actin binding
[12,13,17,20] and perhaps severing [45]. The manner in
which cofilin achieves this feat remains contentious and two
broad models have been proposed. Both propose a binding
geometry where cofilin binds one actin monomer at
subdomain 1, and a second, longitudinally associated
monomer immediately toward the barbed end at subdo-
main 2. The major difference between the models is in the
Fig. 5. Competition binding study between actin fragment 84–103 and
G-actin monitored by ELISA. The binding of cofilin (1.1 l
M
)tocoated
actin peptide of sequence 360–372 in 50 m
M
Mops buffer pH 7.5 (s)
or 6.6 (d), supplemented with 3% gelatin hydrolysate, in the presence
of increasing G-actin concentrations (0–2.4 l
M
). Binding was detected

by using anti-cofilin Ig and monitored at 405 nm.
Fig. 6. Binding of cofilin with 84–103 actin sequence evidenced by
fluorescence. Changes in the emission spectrum intensities of 84–103
peptide (0.6 l
M
) labeled with Oregon green were monitored in the
presence of cofilin (0–7 l
M
). The experiments were carried out in
50 m
M
MOPS buffer pH 6.5 (d)orpH8.0(s).
Fig. 7. Interaction of RITC-labeled cofilin with 84–103 actin sequence
evidenced by fluorescence. Changes in the emission spectrum intensities
of RITC-cofilin (2 l
M
) were measured in the presence of 84–103
peptide (0–20 l
M
). The experiments were carried out in 50 m
M
Mops
buffer pH 6.5 (d)orpH8.0(s).
Fig. 8. Binding of purified antibodies directed to 75–105 sequence of
actin to G-actin monitored by fluorescence measurements. Changes in
the emission spectrum intensities of antibodies (0.25 l
M
)labeledwith
Oregon green were monitored in the presence of G-actin (0–4.2 l
M

).
The experiments were carried out in 50 m
M
Mops buffer pH 6.5 (d)or
pH 8.0 (s).
4198 L. Blondin et al.(Eur. J. Biochem. 269) Ó FEBS 2002
position of the cofilin with respect to the second associated
monomer. We [32,33] propose that cofilin intercalates into
the filament between the two associated monomers to bind
the second through an interaction with the upper ÔrearÕ of
subdomain 2. A number of other groups [20,31,46], suggest
that cofilin binds the ÔforwardÕ facing surface of subdomain
2 (that is, in the standard orientation as first displayed by
Kabsch and colleagues [47]). These models predict profound
differences in the surfaces of cofilin that would be exposed at
the surface of the cofilin:actin filament, and in the interfaces
between the molecules. An additional complexity is that in
one reconstruction, a second ADF/cofilin was proposed to
bind the filament [31] so that the over all ratio in these
filaments was two ADF/cofilins to every actin, this would
perhaps explain why others have found bundling activity
associated with ADF/cofilins [48]. However, both pheno-
menon could be explained by oxidation of the many
cysteine residues carried by these proteins.
The interaction of typical ADF/cofilins is pH sensitive
but the molecular mechanism has not yet been explained.
The pH sensitivity could result from three nonexclusive
possibilities: it may arise from titratable residues on either
surface; alternatively, the tertiary structure of cofilin may
undergo a pH-sensitive change, or finally, a conformational

change could be displayed by actin, either by actin
monomers or between monomers associated within the
filament.
Binding of cofilin to G-actin at site 1 is pH-insensitive
The ADF/cofilin family bind G-actin through subdomain 1
[49,50]. We have shown here by two independent means that
the interaction of ADF/cofilin with G-actin through site 1 is
not sensitive to pH within normal physiological range
(pH 6.5–8.0). We measured the affinity of actin and cofilin
by changes in the fluorescence of RITC labelled actin.
Although the fluorescence change between RITC-actin and
cofilin was larger when measured at pH 6.5 than at pH 7.8
(Fig. 2A) the calculated affinities were similar (K
d
¼ 1 l
M
).
This value is higher than that typically measured for actin-
G–actin interaction at 0.1 l
M
, probably as a result of the
label as it is known that modification of Cys374 by other
agents inhibits the interaction with cofilin [19]. We measured
the affinity of binding of cofilin to unmodified ADP-G-actin
and ATP-G-actin as a function of pH by direct means in
order to confirm that the lack of pH sensitivity was not an
artifact of labelled actin. We found no difference in binding
of either ADP or ATP-actin to cofilin immobilized on beads
between pH 6.5 and pH 8.0 (Fig. 2B). The G-actin binding
footprint of yeast cofilin has been determined at pH 8.0 by

synchrotron protein footprinting [51]. The G-actin binding
footprint in surprisingly large, encompassing roughly a third
of the surface of cofilin.
No evidence for pH dependent conformational changes
in the structure of cofilin
We could find no evidence for substantial pH-dependent
conformational changes in cofilin that might explain the
pH-dependent nature of the interaction of ADF/cofilins
with F-actin. We measured the intrinsic fluorescence of
cofilin via its unique tryptophan residue and the extrinsic
fluorescence of RITC covalently linked to cofilin at various
pHs between 6.5 and 8.0 and observed no significant
changes in fluorescence intensity in agreement with other
studies using circular dichroism and limited proteolysis [52].
No evidence for pH-sensitivity in the actin:cofilin
surfaces directly
None of the actin-derived peptides that we have previously
showntobindactin,dosoinapH-sensitivemanner.
However, actin itself is pH-sensitive, the spontaneous
polymerization of actin is more rapid at pH 6.5 than at
pH 8.0 [22,53], the intermonomeric flexibility of Mg
2+
-actin
filaments is larger at pH 7.4 than at pH 6.5 [54], and actin
filaments are stabilized at low pH [55].
PH-dependence may result from conformational
changes in the actin monomer itself
We have detected a pH-sensitive change in the structure of
actin subdomain 1 that may explain the overt pH sensitivity
of the cofilin-F–actin interaction. The interaction of Oregon

green coupled antibodies directed to residues 75–105 of
actin is strongly pH-sensitive, most probably because of a
difference in conformation of G-actin at the two pH
extremes. This finding was confirmed by fluorescence
measurements of a similar peptide 83–103 labelled with
Oregon green that again showed pH-dependent changes in
the presence of cofilin. Evidence for a pH sensitive change in
conformation in subdomain 1 of G-actin has come from
studies with AEDANS labeled actin [23]. Residues 75–105
encompass part of cofilin binding site 2 [33] and is situated
between subdomains 1 and 2. FRET analysis has shown
that cofilin binding alters the orientation of subdomain 1
and 2 of actin [33]. We have proposed that cofilin binds a
second site (site 2) on F-actin [32], consisting of a helical
region 112–125 that lies on the Ôupper, rearÕ surface of
subdomain 1 close to subdomains 2 when viewed in the
standard actin orientation [47]. This second site of actin is
proposed to be cryptic, pH sensitive movements of region
75–105, may make site 2 (in region 112–125) available for
binding probably by the C-terminal helix of ADF/cofilin
[46].
We have previously shown that FITC-cofilin binds to
both G- and F-actin and that this induces an increase in
fluorescence in conditions that allow actin oligomerization
to occur [33]. This increase in fluorescence is very much
more rapid than polymerization and probably reflects a
conformational change. We now show that this conforma-
tional change only occurs at pH 6.5 and not at pH 8.0
(Fig. 1), suggesting that site 2 is present only in F-actin and
in G-actin bound through site 1 by cofilin at pH 6.5.

Three actin-binding sites of cofilin?
Present evidence suggests that cofilin binds actin at site 1
through the N-terminal region [49], and site 2 possibly
through the C-terminal helix [46]. Many studies have
indicated that the so called long helix is important to
actin-binding and additionally mutations here dissociate
severing from pointed end off rate increase. It has been
suggested that cofilin binds a third site on actin by a region
around K114 on cofilin’s long helix [56]. This site may be an
as yet unrecognized distinct region on actin, or a region
Ó FEBS 2002 pH-dependence of cofilin–actin interaction (Eur. J. Biochem. 269) 4199
contiguous with those surfaces already identified as sites 1 or
2. We favour the latter hypothesis, and since K114 appears
at the surface of cofilin so close to the N-terminus, we
further hypothesize that site 1 is contiguous with site 3, in
agreement with data obtained by synchrotron protein
footprinting [51]. This is also in agreement with the finding
that a peptide including K114 can be crosslinked to Cys374
on actin [57].
Implications for other actin-binding proteins
The ADF homology domain (ADF-H) is defined as a
protein sequence motif shared between the AC family
members and a number of other proteins distinct from the
ACs [58]. These include twinfilin, which has tandem ADF-
H domains [59], coactosin and Abp1p (see [60]). Surpris-
ingly, the gelsolin repeat is similar to the ADF-H fold
despite having little sequence homology [61]. However
gelsolin domain 2 binds actin through an interface distinct
from that of gelsolin domain1 and both through interfaces
distinct from cofilin [32]. Thus, the group of proteins that

possess ADF-H sequence motifs or that share homologous
folds, tend to share some actin binding properties such as
PIP
2
sensitivity, ADP-actin monomer preference and (in
some cases) pH dependence, yet paradoxically bind actin
through distinct interfaces [62]. Actophorin and depactin
(from Acanthamoeba and star fish eggs, respectively) are
members of the ADF/cofilin family that are not pH
sensitive. Depactin is reported as being not pH sensitive
[16] and actophorin binds to F-actin at both pH 6.5 and
pH 8.0 [17].
Any explanation of pH sensitivity of the ADF/cofilins
must also explain why these otherwise typical ADF/cofilins
are not pH sensitive. It is possible that actophorin does not
share typical pH dependence of F-actin binding because site
2 is not hidden from binding as it is in the case of cofilin.
These experiments are in progress.
ACKNOWLEDGEMENTS
This research was supported by grants from AFM and Amoebics Ltd.
Edinburgh.
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