Binding of gelsolin domain 2 to actin
An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin
Celine Renoult
1
, Laurence Blondin
1
, Abdellatif Fattoum
2
, Diane Ternent
3
, Sutherland K. Maciver
3
,
Fabrice Raynaud
1
, Yves Benyamin
1
and Claude Roustan
1
1
UMR 5539 (CNRS) Laboratoire de Motilite
´
Cellulaire (Ecole Pratique des Hautes Etudes), Universite
´
de Montpellier, France;
2
Centre de Recherches de Biochimie Macromole
´
culaire, Montpellier, France;
3
Genes and Development Group,

circulation [4], while the other form is intracellular. In vitro,
gelsolin interacts with G- and F-actins, promotes nucleation
and both severs and caps actin filaments. Cofilin belongs to
another family of actin-binding proteins that also severs
actin filaments and increases polymerization dynamics [5].
Despite a lack of sequence homology between the cofilin
and gelsolin families the fold adopted by each of gelsolin’s
130 amino-acid subdomains [2] is similar to the actin
depolymerizing factor (ADF)/cofilin family fold [6]. In
contrast with cofilin, gelsolin does not appear to be essential
for viability in the organisms where this has been tested,
probably due to the expression of related genes such as
adseverin/scinderin [7], but gelsolin is specifically required
for rapid movement of various dynamic cells [8]. Thus,
gelsolin over-expression in fibroblasts leads to enhanced cell
motility [9,10].
Domains 1–3 (S1–3) are sufficient for capping and
severing, while the C-terminal half of the molecule is
directly implicated in calcium regulation. In particular,
gelsolin domain 1 (S1) interacts both with monomeric actin,
and with the barbed end of the actin filaments inhibiting
polymerization.
S2, in contrast, preferably binds to the side of the actin
filament. Severing activity seems to require the binding of
S2 to the filament, followed by interaction of S1 between
two adjacent actins along the filament axis [11].
The tertiary structure of whole gelsolin in the inactive
Ca
21
free state has been determined [2], as has S1 in

assay; FITC, fluorescein 5-isothiocyanate; G-actin, monomeric actin;
F-actin, filamentous actin; EEDQ, N-ethoxycarbonyl-2-ethoxy-
1,2-dihydroquinoline.
Eur. J. Biochem. 268, 6165–6175 (2001) q FEBS 2001
determination of the S4–6 actin structure. They suggested
that changes in the structure of S1 –3 must occur to allow S2
to interact with the side of actin filament. Finally from
mutagenesis and structural data, Puius et al. [14] proposed a
model for S2 interaction in which 168RRV170 and 210RLK
212 are determinant in F-actin binding.
In this report, we investigated the gelsolin S2 : actin
interface. In particular, we focused on the comparison
between respective locations of gelsolin and cofilin on actin
filament and evidenced major differences in the interfaces.
MATERIALS AND METHODS
Proteins and peptides
Rabbit skeletal muscle actin was isolated from acetone
powder [18], and stored in buffer G (2 m
M Tris, 0.1 mM
CaCl
2
0.1 mM ATP pH 7.5). Actin was selectively cleaved
by Staphylcoccus aureus V8 protease [19] and thrombin
[20] and the fragments obtained were isolated by
electroelution as described previously [19]. Human gelsolin
domain 2 (S2) was produced in Escherichia coli,
BL21(pLysS) carrying a vector containing a cDNA encod-
ing residues including 151 – 266, the S2 repeat [21]. The
bacteria were grown in 1-L flasks with 2 Â TY medium with
ampicillin (150 mg

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 labelled at the cysteine residue with
N-iodoacetyl-N
0
-(sulfo-1-naphthyl)-ethylenediamine (1,5-I-
AEDANS) or at amino groups by fluorescein 5-isothio-
cyanate (FITC) [24,25]. Excess reagent was eliminated
by sieving through a Biogel P2 column equilibrated with
0.05
M NH
4
HCO
3
buffer pH 8.0. Actin and gelsolin S2
domain were labelled by FITC as described elsewhere [25].
Excess reagent was eliminated by chromatography on a
PD10 column (Pharmacia) in 0.1
M NaHCO
3
buffer pH 8.6.
Actin was specifically labelled at cysteine 374 by 1,5-I-
AEDANS [24].
Cross-linking experiments
Actin (1 mg
:
mL
21

2
CO
3
pH 9.5, were immobilized on plastic
microtiter wells. The plate was then saturated with 0.5%
gelatin/3% gelatin hydrolysate in 140 m
M NaCl/50 mM
Tris buffer pH 7.5. Experiments with coated peptides
were performed in 0.15
M NaCl, 10 mM phosphate
pH 7.5. Binding was monitored at 405 nm using alkaline
phosphatase-labelled anti-IgG antibodies (1 : 1000) or
alkaline phosphatase-labelled streptavidin (1 : 1000). Con-
trol assays were carried out in wells saturated with gelatin
and gelatin hydrolysate used alone. Each assay was con-
ducted in triplicate and the mean value plotted after sub-
traction of nonspecific absorption. The binding parameters
(apparent dissociation constant K
d
and the maximal binding
A
max
) were determined by nonlinear fitting A ¼ A
max
[L]/
(K
d
1 [L]) where A is the absorbance at 405 nm and [L] is
the ligand concentration, by using the
CURVE FIT software

d
(apparent dissociation
constant) and A
max
(maximum effect) were calculated by
nonlinear fitting of the experimental data points.
The number of binding sited (n ) and the affinity constant
K
a
were also determined by another approach [29,30]. The
6166 C. Renoult et al. (Eur. J. Biochem. 268) q FEBS 2001
following relationship was then used:
1/ð1 2 XÞ¼K
a
ðC/ ðXEÞ 2 nÞð1Þ
where C and E are total concentrations of peptide and actin,
respectively, and X is the relative fluorescence change
A/A
max
(corresponding to the fraction of peptide bound to
actin).
A plot of 1/(1 – X) ¼ vs. C/(XE) (Eqn 1) was drawn. The
plot gives the number of binding sites which is the value
of C/(XE) for 1/(1–X) ¼ 0. The slope of the same curve
directly gives the value of the affinity constant.
Analytical methods
Protein concentrations were determined by UV absorbency
using a Varian MS 100 spectrophotometer. Electrophoresis
was carried out on 12.5% (w/v) polyacrylamide slab gels
(SDS/PAGE 12.5%) according to Laemmli [31] and stained

environment of Cys374 occurring during actin–peptide
complex formation. In a second approach, FITC-labelled
actin was incubated in the presence of increasing
concentrations of 203 – 225 peptide (0–19 m
M). The results
shown in Fig. 1 indicate change in the FITC fluorescence
induced by complex formation. The shape of the curve
shows that the binding takes place in a saturable manner
with an apparent K
d
of 5 mM. These experiments confirm the
results of van Troys et al. [33] which implicate the sequence
197–226 in the gelsolin : actin interface.
A second gelsolin S2 : actin interface is located in the
N-terminal part of S2. In order to delimit the footprint of this
gelsolin part on the actin structure, a peptide covering the
159–193 sequence was synthesized. Its conformation in an
aqueous solution was studied by IR in the amide 1 region.
The second derivative of the spectrum (Fig. 2), character-
ized by a major band at 1629
:
cm
21
associated with a band at
1680
:
cm
21
suggests the presence of an antiparallel beta
sheet structure [36]. In the corresponding region of gelsolin

experiments. G-actin labelled with FITC was incubated in
the presence of increasing 159– 193 peptide concentration
and the changes in fluorescence were monitored. The
saturation curve observed suggests a specific interaction
with a K
d
of 2 mM. A stoichiometry of < 1 mole peptide per
mole G-actin was also estimated (Fig. 4C). A similar
experiment was performed with dansylated F-actin at
Cys374 (Fig. 4B inset). The interaction induces a
fluorescence quenching of the chromophore (K
d
¼ 2 mM).
Determination of the 159–193 peptide/actin interface
Two approaches were used for identification of large
fragments of actin to which gelsolin 159–193 peptide could
be cross-linked by EEDQ. They involved the electrophoretic
and immunological analysis of the cross-linked products
formed either on proteolysis of the complex by V8 protease
or on cross-linking of the 159–193 peptide to actin after
digestion by thrombin. Digestion of actin by V8 protease
gives two major fragments [19] of 31 and 16 kDa (1 – 225
and 226–375 sequence, respectively). As shown in Fig. 5,
digestion of the cross-linked actin peptide complex reveals
two faint bands at 33 kDa and 46 kDa which are missing
from the controls. They can be stained by both anti-actin
(directed towards sequence 75–105) and anti-gelsolin
Fig. 4. Binding of gelsolin fragment 159 –193 with actin. (A)
Interaction of gelsolin fragment monitored by ELISA. Coated G-actin
was reacted with the gelsolin fragment at the concentrations indicated.

band resulting from the covalent association between the
27 kDa fragment of actin (114–375 sequence) and the
gelsolin fragment. This conclusion is supported by the fact
that this band can be revealed by anti-gelsolin and anti-actin
antibodies (directed towards 285–375 sequence) (Fig. 6)
These results reveal that the cross-linking reactions impli-
cate the residues within the 114–225 sequence of actin.
Two large purified actin fragments [19,20] derived
from thrombic and V8 protease digestion of actin
(114–375 and 226 – 375 fragments) were tested for their
possible interaction with 159–193 peptide by ELISA. The
results shown in Fig. 7 indicate that both large fragments
interacted with the gelsolin peptide. However binding to
the 114–375 fragment was of higher affinity (apparent
K
d
¼ 1.8 mM) that binding to the 226–375 fragment
(apparent K
d
¼ 10 mM). Therefore, these results locate the
actin site in central and C-terminal parts of actin.
Identification of amino acid sequences implicated in the
interfaces between actin and 159–193 fragment
In the N-terminal extremity, the sequence 18–28 was
previously show to be involved in gelsolin S2–3 domains
binding [38]. We tested here the ability of the sequence to
interact with the 159 –193 peptide. ELISA experiments in
which 18–28 peptide was coated to plastic showed no
Fig. 5. Analysis of the cross-linking between the gelsolin fragment 159–193 and actin with EEDQ after protease V8 digestion. The cross-
linking reactions followed by a limited digestion by the V8 protease were carried out as described in Material and methods. Proteolysed material was

119–132 peptide does not perturb FITC. In contrast
the 112–125 peptide induces a fluorescence decrease of
the label, but the corresponding binding is very weak
(K
d
. 50 mM). Therefore, to test the 119–132 sequence,
corresponding peptide was synthesized with an extra
cysteine at the N-terminal extremity, then labelled with
1,5-I-AEDANS. The binding of the gelsolin fragment
increases the dansyl fluorescence (Fig. 8C). Analysis of the
saturation curve shows binding parameters which confirm
the ELISA results (K
d
¼ 2 mM).
A second interface was then evidenced in the C-terminal
part of actin. The more accessible sequences in this region
were first investigated by ELISA. One corresponds to the
helix 338–348, and the other to two helices and one turn
located in the 356–375 sequence. The corresponding
peptides (339–349, 347–365, 356–375 and 360–372) were
coated to plastic. We observed (Fig. 8A and Table 1) that
only peptides 356–375 and 347–365 interacted signi-
ficantly with the gelsolin fragment. The activities of
overlapping peptides within the C-terminal of actin towards
Fig. 7. Interaction of gelsolin peptide 159–193 with two large
C-terminal fragments of actin. Actin (0.5 mg
:
mL
21
)(W) or two actin

gelsolin fragment were added to peptide 119–132 (X), 347–365 (W)
and 360–372 (B) in 0.05
M Tris buffer pH 7.6.
6170 C. Renoult et al. (Eur. J. Biochem. 268) q FEBS 2001
FITC-labelled gelsolin fragment were finally tested by
fluorescence. As shown in Fig. 8B, only 356 –375 peptide
interaction can be characterized by this method. Finally,
dansylated peptides 347–365 and 360–372 were tested. The
peptide interaction of 348–365 peptide with the gelsolin
fragment was evidenced (Fig. 8C). All of these facts
suggested corresponding interfaces to be located in the
C-terminal part of actin.
Competition between the N-terminal part of gelsolin S2
and cofilin
Van Troys and colleagues [16] have proposed that cofilin
and gelsolin S2 share a similar target site on the filament. To
show the overlapping of these two proteins on the actin
surface, competition between cofilin and the gelsolin
159–193 fragment was studied by ELISA. G-actin was
coated to plastic and increasing concentrations of gelsolin
peptide were added to a fixed concentration of cofilin
(0.8 m
M). The binding of the ligand used at a fixed con-
centration was monitored using the corresponding cofilin-
specific antibodies. The results presented in Fig. 9 indicate
that a ternary complex actin–cofilin–gelsolin peptide might
occur as the binding of cofilin decreases only partially to
< 45% as the gelsolin peptide concentration is increased.
Footprint of gelsolin S2 on actin
To confirm the ability of sequences 119–132, 18 –28 and

mented with 1% BSA and 0.1 m
M dithiothreitol was performed in the
presence of increasing gelsolin fragment concentrations (0–24 m
M).
Binding was detected by using anti-cofilin antibodies and was
monitored at 405 nm.
q FEBS 2001 The actin gelsolin domain22 interface (Eur. J. Biochem. 268) 6171
actin were added. As shown in Fig. 10A, we observed
changes in the fluorescence intensity. Analysis of these data
give an apparent K
d
of < 5 mM. The interactions evidenced
for gelsolin domain 2 with the three actin peptides (peptides
18–28, 119 –132 and 356–375 [39]) labelled, either with
dansyl or FITC (Fig. 10B), are in agreement with the above
results.
Finally competitions for the binding of S2 and actin
peptides to G-actin were also performed (Fig. 10C). We
observed the dissociation of actin–S2 complex by peptides
119–132 and 356–375. However peptides 18–28 and
338–348 had no effect.
DISCUSSION
The actin-binding site on S2
S2 (137–247) contains gelsolin’s initial F-actin binding
site prior to severing/capping microfilaments [40], but the
orientation of contacting residues and to a lesser extent
the identity of these residues within S2 is less certain. The
first 10 residues of S2 in addition to S1 is the minimal
requirement for filament severing [41]. The standard
explanation for this is that a very weak F-actin binding

and short length of peptide 203–225 has made the deter-
mination of its binding site on actin and the stochiometry of
Fig. 11. A model for the interaction of gelsolin with the actin
filament. Our data suggest that S1 and S2 bind to the same actin
monomer exposed at the barbed end of the filament after severing. S3
acts as a spacer connecting S2 to S4 which binds either to the diagonally
opposed actin monomer ‘a’ or monomer ‘b’. We prefer monomer ‘a’ as
this affords the shortest distance across the filament. S4 binds the actin
monomer with a similar interface as S1. S5 and S6 do not, as far as is
known, bind actin and may stick out from the filament as illustrated.
Table 1. Summary of binding of gelsolin peptide 159–193 and cofilin to various parts of actin and whole actin in the F- and G-form by similar
methods. Note that no K
d
value is given for cofilin binding to F-actin as the co-operativity of the interactions precludes this. ND, Not determined.
Tested
sequences
Peptide
159–193
K
d
ELISA
Peptide
159–193
K
d
fluorescence
Cofilin
K
d
Reference

Van Troy and colleagues [33] have used sequence-specific
actin antibodies to localize the site cross-linked to S2
peptide 198–227 and found that they could exclude residues
12–44, 228–257 and 354–375 from being the site of
peptide binding. Our adjacent peptide S2 159–193 did not
bind the C terminus of actin (360–372) either but we did
measure a reasonable binding (K
d
3–5 mM) to actin peptide
355–375 and to 347–365 (K
d
2 mM) (Table 1). It is possible
that both S2 and the antibody used by this group [33] are
able to bind 355–375 of actin simultaneously. We measured
tight binding (K
d
1.8 mM) to 114–375 and weaker binding
(K
d
10 mM) to 226–375 of actin. As the affinity for S2 to the
entire actin molecule is within this range (K
d
1.4–7.9 mM)
[14,32] perhaps there is no other region on the surface of the
actin molecule that binds actin. This is not compatible with
Puius et al. [14] who postulated a second monomer interface
with the DNase1 binding loop of actin in subdomain 2. Pope
et al. [44] have shown that DNase1 does not interfere with
the binding of S2–3, but perhaps binding can occur through
the first actin binding site in S2 [14].

similarity and the fact that both gelsolin and ADF/cofilin are
actin-binding proteins, the fold seems to form at least three
quite distinct actin binding interfaces. Ultimately, structural
solutions of both S2-decorated and ADF/cofilin-decorated
F-actin will be required to establish the exact F-actin
binding characteristics of these different protein families
and how similar or otherwise they truly are.
The orientation of S2 with respect to actin, and
implications for gelsolin on the microfilamen
t
How the six gelsolin domains arrange themselves around
the actin filament to sever and cap it remain controversial.
We have characterized an S2-binding site on subdomain 1
of actin adjacent to but not overlapping that of the S1 site
between subdomains 1 and 3 [11]. S1 plus a short peptide
(Phe134–Gln160) running into S2 is sufficient for severing
[41]. As this is likely to be brought about by weak F-actin
binding by the peptide, and this region is so close to S1 it is
probable that the N terminus of S2 binds subdomain1 of
actin. We now report that 159–193 of S2 binds to regions
within 119 –132 and 347 –375 of actin both towards the
outer surface of the filament on subdomain 1. The actin
monomer is generally flat, and in the standard orientation
the actin monomer has it flat face presented. We have
determined that S2 binds subdomain 1 on the lower edge
and even perhaps ‘behind’ this flat face surface. This
placement would explain the capping activity observed in
S2 [32] as binding in this region would prevent monomer
addition at the barbed end by blocking the longitudinal
binding site between subdomain 1 and the DNase1 site of

connects S2 to S3 by
wrapping around S1. The position of S2 at the edge of
subdomain 1 shortens the distance that S3 has to straddle S2
and S4.
Major rearrangements between the domains must occur
between the Ca
21
-free and Ca
21
-bound gelsolin [2,12].
There is presently little data to distinguish if S4, which binds
actin [56] in a manner to S1 [12], binds the actin monomer
(a) as shown (Fig. 11) or the monomer that would have been
q FEBS 2001 The actin gelsolin domain22 interface (Eur. J. Biochem. 268) 6173
placed immediately under it (b); however, we prefer the
model as shown as it seems that this would be the shortest
route given how the backbone is positioned at the C terminus
of S2. The positions of S5 and S6 relative to the capped
filament are not known with any precision but are shown
‘sticking out’ from the filament as electron microscopic data
from gelsolin S2–6-decorated microfilaments [17] indicate
that this is possible.
ACKNOWLEDGEMENT
We thank P. McLaughlin for many valuable comments on the work.
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