articles
Interaction of WASP/Scar proteins
with actin and vertebrate Arp2/3
complex
Jean-Baptiste Marchand*, Donald A. Kaiser*, Thomas D. Pollard*† and Henry N. Higgs*
*Structural Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA
†e-mail: [email protected]
The Wiskott–Aldrich-syndrome protein (WASP) regulates polymerization of actin by the Arp2/3 complex. Here we
show, using fluorescence anisotropy assays, that the carboxy-terminal WA domain of WASP binds to a single actin
monomer with a Kd of 0.6 µM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences con–1
tribute to actin binding. A favourable ∆H of –10 kcal mol drives binding. The WA domain binds to the Arp2/3
complex with a Kd of 0.9 µM; both the C and A sequences contribute to binding to the Arp2/3 complex.
Wiskott–Aldrich-syndrome mutations in the WA domain that alter nucleation by the Arp2/3 complex over a tenfold
range without affecting affinity for actin or the Arp2/3 complex indicate that there may be an activation step in
the nucleation pathway. Actin filaments stimulate nucleation by producing a fivefold increase in the affinity of
WASP-WA for the Arp2/3 complex.
he Arp2/3 complex1 initiates actin-filament assembly in motile
cells by nucleating filaments that grow in the barbed direction
as branches from existing filaments2,3. The Arp2/3 complex is
concentrated at the leading edge of motile cells4–6 and is localized to
filament branches7. The complex is intrinsically inactive in nucleation and branching, and therefore requires activation8. The first
activator to be identified was ActA9, a surface protein of the bacterium Listeria monocytogenes that is necessary and sufficient for actinbased motility of the bacterium in the cytoplasm of eukaryotic cells.
The first recognized eukaryotic activator of the Arp2/3 complex was
the C terminus (WA domain) of WASP/Scar family of proteins,
which consists of a WH-2 motif (W) that binds to actin monomers
and an acidic tail (A) that binds to the Arp2/3 complex10–14. A cofilin-homology motif has been postulated between the WH-2 and A
sequences, but the structure of these residues bound to the GTPasebinding domain (GBD) of WASP15 is different to the corresponding
sequence in ADF/cofilins16,17, so we refer to it as the connecting
sequence, C. Actin filaments enhance WA-mediated activation of
nucleation by the Arp2/3 complex8,11. Together, WASP/Scar WA and
actin filaments can stimulate every Arp2/3 complex in an assay to
create a new actin filament, largely as branches3.
WASP is the product of the gene that is mutated in human
patients with Wiskott–Aldrich syndrome, a disease that causes variable defects in blood clotting and immune defences as a result of
deficiencies of the actin cytoskeleton of white blood cells and
platelets18. Cells in other tissues express proteins related to WASP
called N-WASP and Scar isoforms1–3. All have C-terminal WA
sequences that activate the Arp2/3 complex11,12,14, but each
WASP/Scar protein differs in the regulatory domains present in the
amino-terminal 85% of these molecules. WASP and N-WASP each
have an EVH1 domain that binds to proline-rich sequences19,20, and
a GBD that binds either to the Rho-family GTPase Cdc42 (ref. 21) or
to the C region of the WA domain15. Interaction of the GBD with the
C region auto-inhibits the ability of WA to activate the Arp2/3 complex22. Cdc42 and polyphosphoinositides relieve this auto-inhibition,
although the literature is quite divergent on the mechanism12,22–24.
Here we characterize interactions of WASP-WA and Scar-WA
with actin monomers and the Arp2/3 complex to learn how WA
activates actin nucleation by the Arp2/3 complex and how mutations in WA cause disease in Wiskott–Aldrich-syndrome patients.
T
76
We show that efficient actin nucleation requires both recruitment
of an actin monomer to the Arp2/3 complex and a subsequent activation step. We also show that the first two steps in this pathway,
binding of WA to an actin monomer and to the Arp2/3 complex,
are both rapidly reversible equilibria with submicromolar affinities,
with binding to actin favoured over binding to the Arp2/3 complex.
Actin filaments seem to stimulate nucleation by enhancing binding
of WA to the Arp2/3 complex.
Results
Characterization of rhodamine–WASP-WA. We used fluorescence
anisotropy assays to measure binding of WASP-WA labelled with
tetramethylrhodamine-5′-maleimide to actin and the Arp2/3 complex. We used similar assays to study interactions of profilin25 and
actophorin26,27 with actin monomers and filaments. The N-terminal border of the WA domain is not rigorously defined, so we evaluated four WASP-WA constructs of different lengths (Fig. 1a).
WASP-WA does not contain cysteine, so we added a cysteine
residue to the N terminus of each construct. Fluorescence
anisotropy of rhodamine–WASP-WA varied between constructs
and preparations from 0.08–0.10, as expected for peptides of relative molecular mass (Mr) 8,600–10,600.
Binding of WASP-WA to actin. Titration of the four rhodamine–WASP-WA constructs with muscle Mg-ATP–actin
monomers in polymerization buffer yielded binding isotherms
(Fig. 2a) with Kd values of 0.05–0.35 µM (Table 1). Sedimentationequilibrium ultracentrifugation of 9 µM WASP-429-WA (Fig. 1a)
with 20 µM actin and 40 µM latrunculin B in KMEI buffer (10 mM
imidazole pH 7.0, 50 mM KCl, 1 mM EGTA and 1 mM MgCl2)
gave an Mr of 51,000 (51K) for the rhodamine (measured at
550 nm), which corresponds to the Mr of a 1:1 complex of WA with
an actin monomer. The affinity of rhodamine–WASP-406-WA for
Mg-ADP–actin (Kd 1.2 µM) was threefold lower than for MgATP–actin. Fluorescence anisotropy of rhodamine–WASP-406-WA
was the same with or without 5 µM polymerized actin (Fig. 2d),
confirming that it does not bind to actin filaments8,10.
Competition between unlabelled WASP-429-WA and each
labelled WASP-WA construct for binding to actin (Fig. 2b) yielded
a Kd of 0.6 µM for unlabelled WASP-429-WA (Table 1), the same as
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articles
a
A
C
CA
W
WASP-429-WA
WASP-427-WA
WASP-424-WA
WASP-406-WA
Scar1-445-WA
* **
**
*
*
SGNGPAPPPLPPALVPAGGLAPGGGRGALLDQIRQGIQLNKTPGAPESSALQPPPQSSEGLVGALMHVMQKRSRAIHSSDEGEDQAGDEDEDDEWDD
----------------------SDARSVLLEAIRKGIQLRKVEEQRE---QE---AKHERIENDVATILSRR-IAVEYSDS-EDDS-EFDEVD-WLE
WH2
Central
Acidic
b
?
+
+
?
+
+
Activation
?
+
1,000 s–1
+
Elongation
100 s–1
10 s–1
1 s–1
0.1 s–1
+
Figure 1 WASP constructs and nucleation reactions. a, Sequences and domain
structures of WASP and Scar-1 constructs. Positions of the point mutations introduced in this experiment are indicated in green; those of Wiskott–Aldrich-syndrome
mutations are shown in red. b, Pathway of actin-filament nucleation by the Arp2/3
complex (green and grey). In the pathway favoured by the kinetic constants and
protein concentrations (shaded), actin (yellow) binds to the WA domain (red),
actin–WASP-WA binds to the Arp2/3 complex, and the actin–WASP-WA–Arp2/3
complex binds to the side of an actin filament. Filament binding enhances the interaction of the actin–WASP-WA–Arp2/3 complex, increasing the chance of forming a
stable nucleus. An activation step is postulated to precede growth of the new filament as a branch. Arrows indicate approximate reaction rates under physiological
conditions.
that measured in assays of pointed-end polymerization under these
conditions8. Thus, rhodamine labelling increased the affinity of
WASP-WA for actin, and competition of unlabelled constructs with
rhodamine–WASP-429-WA yielded reliable measurements of affinity.
Human profilin-I and thymosin-β4 competed with rhodamine–WASP-429-WA for binding to actin monomers, indicating
that their actin-binding sites may overlap. Their measured affinities
for actin (Kd 0.17 µM for profilin and 0.8 µM for thymosin) are
consistent with data obtained by other methods25. Latrunculin B
did not affect binding of WASP-WA to actin in polymerization
buffer, and 12 µM latrunculin B completely inhibited polymerization of 4 µM 5% pyrenyl-actin.
Human Scar1-WA competed with rhodamine–WASP-WA for
binding to actin monomers with a Kd of 0.1 µM. As the yields of
recombinant Scar1-WA were higher than those of any WASP-WA
construct, we used Scar1-WA in kinetic and thermodynamic experiments. The time course of displacement of rhodamine–WASP-WA
from actin by Scar1-WA followed a single exponential with a value
of 3 s–1 (Fig. 2c). Using this rate constant and the Kd value, the calculated association rate constant for binding of rhodamine–WASPWA to actin is 43 µM–1 s–1.
Three different methods indicated that neither WASP-WA nor
Scar1-WA has a compact structure. Circular-dichroism spectra had
minima at 200 nm. Deconvolution of these spectra by four methods indicated small fractions of secondary structure: WASP-WA
(1–9% α-helix, 4–13% β-sheet, 65–75% turn and unordered);
Scar1-WA (8–11% α-helix, 7–17% β-sheet, 65–85% turn and
unordered). Sedimentation-velocity ultracentrifugation of Scar1WA in KMEI gave a sedimentation coefficient of 1.0 S, a diffusion
coefficient of 10.2 µm2 s–1 and a frictional ratio of 1.3, which is
indicative of an asymmetric particle. Titration of Scar1-WA with
urea gave a linear twofold increase of intrinsic tryptophan fluorescence rather than a sharp transition, which is typically observed
when compact proteins with buried tryptophan residues unfold.
Titration calorimetry provided an independent method to
assess the stoichiometry and affinity of Scar-WA binding to actin
monomers and a direct test of whether binding involves immobilization of the conformationally flexible WA domain. Titration of
actin with Scar1-WA in polymerization buffer with excess latrunculin B to prevent polymerization (Fig. 2g, h) yielded an equilibrium constant (Kd 0.2 µM) that was similar to the spectroscopic
measurements, a stoichiometry of 1:1 and ∆H of –10.1 kcal mol–1
(see Supplementary Information). None of this actin pelleted in the
ultracentrifuge, confirming that latrunculin B prevented polymerization throughout the long titration in polymerization buffer. In
low-salt buffer, binding of Scar1-WA to actin was tenfold weaker,
but ∆H was similar (–9 kcal mol–1). The temperature dependence
of ∆H in low-salt buffer (Fig. 2i) gave a heat-capacity change of
–315 cal mol-1 K–1. See Discussion for an explanation of how these
thermodynamic parameters show that the conformation of WA is
restricted when it binds to actin.
Mapping of the interaction between WASP-WA and actin. Peptides
(Fig. 1a) corresponding to the W and CA regions, but not C or A, of
WASP competed weakly with rhodamine–WASP-429-WA for binding to monomeric actin (Table 1). Thus, WASP-WA has at least two
independent, low-affinity sites that bind to actin. Actin binding was
further characterized by inducing the following point mutations in
WA: Q443R, N445K and K446T in a QLNK motif that is conserved
among WASP/Scar proteins and WIP/verprolin28; and K476E,
R477K and D485N, three missense mutations that occur in patients
with Wiskott–Aldrich syndrome29–31. In competition against rhodamine–WASP-429-WA, the affinities of WASP-WA(Q443R) and
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articles
a 0.25
d
g
0.2
0.15
0.16
µcal s–1
Anisotrophy
Anisotrophy
0.0
0.2
0.12
–0.2
–0.4
–0.6
0.1
0.1
5
10
15
20
25
30
35
0
0.2
Anisotrophy
f
0.15
0.1
0.05
10
15
20
25
30
Molar ratio (Scar1-WA/actin)
i
0.15
0.1
140
–8
–10
35
0.2
120
0
–6
WASP-429-WA concentration (µM)
c 0.25
Anisotrophy
WASP-429-WA concentration (µM)
5
∆H (kcal mol–1)
0
–4
4.5
0.12
0
–2
4.0
Anisotrophy
Anisotrophy
0.15
0.14
2
3.5
0.16
0.25
0.2
Time (min)
h
1.0
1.5
e
0.5
b
100
1
80
0.8
3.0
0.6
2.5
0.4
60
0.2
Arp2/3 complex concentration (µM)
2.0
0
Actin concentration (µM)
40
2.5
2
0.0
1.5
–0.5
1
kcal per mole of injectant
0.5
–20
0.08
0
20
0.1
–5
–6
–7
–8
–9
–10
–11
–12
15
20
25
30
35
0.05
0
1
2
3
4
5
0
2
4
Time (s)
6
8
10 12 14 16
Temperature (°C)
Time (s)
Figure 2 Kinetics and thermodynamics of binding of the WA domain to actin
monomers and to the Arp2/3 complex. Fluorescence anisotropy assays for
binding of WASP-WA constructs to rabbit-muscle actin monomers (a–c) and to the
bovine Arp2/3 complex (d–f). Conditions were as follows: 10 mM imidazole pH 7.0,
50 mM KCl, 1 mM EGTA and 1 mM MgCl2, 22 °C. a, Equilibrium binding of four different constructs of WASP-WA labelled with tetramethylrhodamine. Rhodaminelabelled WASP-WA constructs (0.1 µM) were titrated with Mg-ATP–actin. Curves are
best fits of equation (1) yielding the equilibrium constants listed in Table 1. Filled triangles, WASP-406-WA; open triangles, WASP-424-WA; circles, WASP-427-WA;
squares, WASP-429-WA. b, Competition between unlabelled WASP-429-WA and
three different rhodamine-labelled WASP-WA constructs for binding to monomeric
actin. Rhodamine–WASP-424-WA (0.1 µM, open triangles) or rhodamine–WASP-429WA (0.1 µM, squares) with 0.5 µM Mg-ATP–actin, or rhodamine–WASP-406-WA
(0.1 µM, filled triangles) with 3 µM Mg-ATP–actin were titrated with WASP-429-WA.
Curves are best fits of equation (2) yielding the equilibrium constants listed in Table
1. c, Time course of dissociation of rhodamine–WASP-429-WA from monomeric
actin. A solution of 0.2 µM rhodamine–WASP-429-WA and 0.4 µM Mg-ATP–actin
was mixed with an equal volume of 10 µM Scar1-WA and anisotropy was recorded
at 40 points per s. The smooth curve is the best single exponential fit to the average of 5 data sets yielding k – = 3 s–1. d, Equilibrium binding of rhodamine–WASP406-WA to the Arp2/3 complex. Rhodamine–WASP-406-WA (0.025 µM) was titrated
with the Arp2/3 complex in the presence (circles) or absence (squares) of 5 µM
actin filaments. Curves are best fits of equation (1) yielding the equilibrium constants listed in Table 1. e, Competition between unlabelled and rhodamine-labelled
WASP-429-WA for binding to the Arp2/3 complex. Rhodamine–WASP-429-WA
(0.025 µM) with 1 µM Arp2/3 complex was titrated with WASP-429-WA. The curve
is the best fit of equation (2) to the data yielding the equilibrium constant in Table 1.
f, Time course of dissociation of rhodamine–WASP-429-WA from the Arp2/3 complex. A solution of 0.2 µM rhodamine–WASP-429-WA and 3 µM Arp2/3 complex
was mixed with an equal volume of 20 µM Scar1-WA. Anisotropy was recorded at
20 points per s. The smooth curve is the best single exponential fit to the average
of 5 data sets yielding k – = 0.2 s –1. g–i, Isothermal titration calorimetry of Scar1WA binding to actin monomers. g, Raw titration data showing the heat changes produced by each addition of Scar1-WA during the time course of the experiment.
Titration was carried out by injecting 5.0 µl of 450 µM Scar1-WA at 2-min intervals
into 1.3 ml of 25 µM monomeric actin, each in 2 mM imidazole pH 7.0, 50 mM
KCl, 3 mM NaN3, 0.5 mM dithiothreitol, 0.2 mM ATP, 0.1 mM CaCl2 and 50 µM
latrunculin B at 26 °C. h, Binding isotherm for the data in g. Values are normalized
integrated areas for each of the peaks shown in g; the curve is the best fit to the
data. For this experiment, n = 1.2, Ka = 4.5 × 106 and ∆H = –9.289 kcal mol–1. i,
Temperature dependence of ∆H for binding of Scar1-WA to monomeric actin in
buffer G (2 mM imidazole pH 7.0, 0.5 mM dithiothreitol, 0.2 mM ATP, 0.1 mM CaCl2
and 3 mM NaN3). The slope of a linear fit through these points is the heat-capacity
change, ∆Cp = –315 cal K–1.
WASP-WA(N445K) for actin were twofold and fourfold higher
than the wild-type, respectively, whereas the affinity of WASPWA(K446T) was threefold lower (Table 1). All three WAS mutant
proteins had the same affinity for actin as wild-type WASP-WA.
Comparison of the affinities of these mutant constructs with their
abilities to stimulate actin nucleation revealed the existence of an
activation step subsequent to binding (see Discussion).
Binding of WASP-WA to the Arp2/3 complex. The fluorescence
anisotropy of rhodamine–WASP-429-WA bound to the Arp2/3
complex (0.18) was lower than for saturating actin (0.23, Fig. 2a, d)
even though the Arp2/3 complex is five times larger. Rhodamine
may be less immobilized when bound to the Arp2/3 complex than
to actin. The Kd values of rhodamine–WASP-429-WA for two different preparations of the Arp2/3 complex were 0.22 and 0.28 µM,
78
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Table 1 Interaction parameters of WASP-WA constructs
Equilibrium constant for
actin–ATP (µM)
Equilibrium constant for
the Arp2/3 complex (µM)
Arp2/3 complex activation
normalized to WASP-429-WA*
Labelled,
direct
binding
Unlabelled, competition
with rhodamine–
WASP-429-WA
Labelled,
direct
binding
Unlabelled, competition
with rhodamine–
WASP-429-WA
Labelled
429-WA
0.07†
0.6†
0.22/0.28
0.92†
0.3
427-WA
0.05
Unlabelled
WASP-WA constructs
424-WA
0.05
406-WA
0.35†
0.3
0.39†
0.3
WASP-WA mutants
Q443R
0.28
1.4
1.3
N445K
0.15
0.9
1.7
K446T
1.7
1.1
1.3
K476E
0.55
0.35
0.89
2
R477K
0.55
0.3
0.84
0.5‡
D485N
0.68
0.45
0.81
1.1
W500S
1.7
No binding
No binding
0.2
WASP-WA peptides
W
5.5
No binding
No activity
C
No binding
No binding
No activity
CA
6
1.8
0.005
A
No binding
9
No activity
*Activation is defined as activator concentration required for half-maximal activation of the Arp2/3 complex, as judged by the concentration of filament ends produced. For WASP-429-WA, this
value is 425 µM.
†
Values are means of two to five independent measurements. s.d. ≤ 10% of the mean. Other Kds presented here were determined in one experiment including at least ten measurements. Curve
fitting using equation (1) gave errors of 10–20% of the equilibrium constant value.
‡
Concentration of filaments made at optimal WASP-WA(R477K) concentration was 50% (4 nM) of that made by WASP-429-WA (8 nM). Therefore, 4 nM was taken as maximal activation for WASPWA(R477K).
and the affinity of rhodamine–WASP-406-WA for the Arp2/3 complex was similar. In competition with rhodamine–WASP-429-WA,
the Kd of unlabelled WASP-429-WA for the Arp2/3 complex was
0.9 µM (Fig. 2e). Thus, as for actin binding, rhodamine increased
the affinity of the WA domain for the Arp2/3 complex, but competition assays yielded reliable measurements of affinity.
The affinity of rhodamine–WASP-429-WA for the Arp2/3 complex was 5–6-fold higher in the presence of 5 µM polymerized actin
(Fig. 2d). Addition of latrunculin B-bound actin monomers to rhodamine–WASP-406-WA saturated with the Arp2/3 complex resulted in a saturable increase in anisotropy, indicating that WASP-WA
can bind to actin and the Arp2/3 complex simultaneously. Neither
actophorin nor profilin competed with rhodamine–WASP-429-WA
for binding to the Arp2/3 complex.
Human Scar1-WA competed with rhodamine–WASP-WA for
binding to the Arp2/3 complex with a Kd of 0.5 µM. Displacement
with unlabelled Scar1-WA (Fig. 2f) gave a rate constant of 0.2 s–1
for dissociation of rhodamine–WASP-429-WA from the Arp2/3
complex and a calculated association rate constant of 0.8 µM–1 s–1.
Mapping of the interaction between WASP-WA and the Arp2/3
complex. Peptides A and CA competed weakly with
rhodamine–WASP-429-WA for binding to the Arp2/3 complex
(Table 1), whereas peptides W and C did not. Although we detected
no interaction of C peptide with the Arp2/3 complex, CA bound to
the Arp2/3 complex with fivefold greater affinity than did A.
Neither the point mutations in the QLNK motif nor the WAS
mutations affected the affinity of WASP-WA for the Arp2/3 complex
(Table 1), but mutation of tryptophan 500 to serine eliminated
detectable binding to the Arp2/3 complex. This tryptophan residue
is conserved in all WASP/Scar-family proteins as well as in fungal
myosin-Is.
Actin-filament nucleation. We used actin-filament nucleation to
assess the ability of WASP-WA constructs to activate the Arp2/3
complex8. The rate of polymerization at every time point in the
assay is directly proportional to the concentration of filament
ends11,32. At 10 nM, Arp2/3-complex nucleation is proportional to
the concentration of activated Arp2/3 complex, which continues to
generate new filaments until actin monomers are depleted8. Under
these conditions the activity of WASP-WA constructs can be measured by comparing the concentrations of WASP-WA constructs
required to activate nucleation by the Arp2/3 complex (Fig. 3a, b).
Unlabelled WASP-429-WA at 0.45 µM activated polymerization
half-maximally. Rhodamine labelling of WA-domain constructs
caused a roughly threefold reduction in their ability to activate the
Arp2/3 complex. None of the W, C or A peptides, either separately
or together, had any effect on nucleation. The activity of CA was
200-fold weaker than that of WA, indicating that stronger actin
binding by WH2 may enhance nucleation by CA.
None of the three mutations in the QLNK motif of WH2 affected the ability of WASP-WA to stimulate actin nucleation by the
Arp2/3 complex, in spite of the fact that these constructs vary tenfold in their affinity for actin. The W500S mutation caused a fivefold reduction in the ability of WASP-WA to activate the Arp2/3
complex, and the wild-type level of activation was not reached even
at high concentrations (Fig. 3b).
The three Wiskott–Aldrich-syndrome mutants differed tenfold
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Fluorescence (arbitrary units)
a
16
12
8
4
0
0
100
200
300
400
Time (s)
b 10
Ends (nM)
8
6
4
2
0
0
2
4
6
8
10
WASP-WA concentration (µM)
Figure 3 Actin-filament nucleation by the Arp2/3 complex activated by
WASP-WA constructs. Mutations R477K and W500S have marked effects on
actin nucleation by the Arp2/3 complex. a, Time course of pyrenyl-actin polymerization. Mg-ATP–actin monomers (4 µM, 5% pyrenyl-labelled) and 10 nM Arp2/3
complex were polymerized in 10 mM imidazole pH 7.0, 50 mM KCl, 1 mM EGTA
and 1 mM MgCl2 at 22 °C, either alone (no symbol) or with 0.5 µM WASP-429-WA
(triangles), 0.5 µM WASP-WA(R477K) (squares), 0.5 µM WASP-WA(W500S) (filled circles), 15 µM WASP-W (filled diamonds), 100 µM WASP-C (open diamonds) or
110 µM WASP-A (open circles). b, Dependence of the number of filaments nucleated (ends) on the concentrations of four WASP-WA constructs. Symbols are as in a.
in their ability to stimulate nucleation, in spite of exhibiting wildtype affinities for actin monomers and the Arp2/3 complex. WASPWA(D485N) showed wild-type nucleation activity, whereas WASPWA(K476E) was twice as effective and WASP-WA(R477K) was
severely defective. Even at high concentrations, WASP-WA(R477K)
did not activate the Arp2/3 complex to the same extent as did the
wild-type WA domain, indicating that this mutant may be defective
in an activation step that is distinct from binding to the Arp2/3 complex or to actin monomers.
Although their ability to activate the Arp2/3 complex was low,
high concentrations of WASP-WA(R477K) or WASP-WA(W500S)
promoted the formation of actin-filament branches, as did the wildtype WA domain (see Supplementary Information, Fig. S1a–d).
WAS mutants may also be defective in their ability to bind to WASPGBD15, but all three WAS mutants bound to WASP-GBD in a pulldown assay (see Supplementary Information, Fig. S1e).
Discussion
Fluorescence anisotropy binding experiments, titration calorimetry
and actin-polymerization assays have yielded many quantitative
insights regarding the interaction of the WA domain of WASP and
Scar with actin monomers and the Arp2/3 complex. Rhodamine
labelling of the WA domain for fluorescence anisotropy increases its
80
affinity for actin monomers and the Arp2/3 complex, but this was
circumvented by competition assays using unlabelled constructs.
The WA domain binds to one actin monomer in an equilibrium
with rapid on/off rates and a Kd of 0.6 µM. This confirms the values for the binding constant measured using inhibition of pointedend elongation by the WA domain8,23. Both the WH-2 and CA
sequences contribute to binding to actin monomers, and the effects
of point mutations indicate that the QLNK motif is important. As
profilin and thymosin-β4 compete with the WA domain for binding to actin, and as WASP-WA–actin behaves like profilin–actin8,23,
the WA-binding site on actin is likely to include domain-1. The lack
of influence of latrunculin B on binding of the WA domain to actin
is consistent with the idea that the binding site for latrunculin A is
between the two lobes of actin33. Rhodamine–WASP-WA dissociates from actin with a rate constant of 3 s–1. The dissociation rate
constant of unlabelled WA domain should be about ten times
faster, as the equilibrium constant of the unlabelled domain is
weaker and it is reasonable to assume that the label does not affect
the association rate constant. Therefore, at equilibrium, WASP-WA
cycles on and off actin monomers on a subsecond time scale.
As most unpolymerized actin in cells is bound to thymosin-β4 or
profilin, WASP-family proteins must compete with these proteins for
the actin monomers required for the Arp2/3 complex to nucleate
new actin filaments. All three ligands bind to ATP–actin monomers
with similar Kd values (0.1–0.8 µM), and all three reactions are rapid
equilibria25,34. Thus, ATP–actin monomers will rapidly equilibrate
among these ligands by mass action. As the concentrations of profilin (25–100 µM) and thymosin-β4 (up to 500 µM in platelets) are
higher than that of WASP (10 µM in neutrophils22), only a small fraction of unpolymerized actin is likely to be bound to WASP.
By several criteria, free Scar-WA constructs are not compactly
folded and binding to actin is driven almost entirely by a favourable
–1
change in enthalpy (∆H = –10 kcal mol ). The entropy change is
unfavourable, in spite of the fact that the negative ∆Cp indicates a
hydrophobic effect, with solvent dissociating from proteins when
they bind. This gives a favourable, positive ∆Ssolvent for the solvent.
The total ∆S = ∆Ssolvent + ∆Sconfig., where ∆Sconfig. is the entropy change
in the proteins. Thus, the entropy change of the WA domain itself
upon binding to actin (∆Sconfig.) must be unfavourable and strongly
negative. Together with the fact that the free WA domain has little
secondary structure, this is strong evidence that it has a more
restricted conformation when bound to actin.
The WA domain binds to the Arp2/3 complex with submicromolar affinity, similar to its affinity for actin, although the two
interactions differ in all other ways tested. Equilibrium exchange of
WASP-WA with the Arp2/3 complex is 50 times slower than with
actin. Assuming that the association rate constants are the same for
labelled and unlabelled WASP-WA, the threefold lower affinity of
unlabelled WASP-WA for the Arp2/3 complex indicates that the
dissociation rate constant is ~0.6 s–1. We did not measure the thermodynamic parameters, but expect, given the paucity of secondary
structure in the free WA domain, that binding to the Arp2/3 complex will also be driven by a change in enthalpy rather than a
change in entropy.
Both the C and A sequences, but not the WH2 peptide, contribute to binding to the Arp2/3 complex. The binding sites for
actin and the Arp2/3 complex in the C region may therfore overlap,
raising the possibility that these two ligands compete for binding.
However, the anisotropy of rhodamine–WASP-WA in an excess of
the Arp2/3 complex is higher when actin is also present, indicating
that the two ligands can bind simultaneously. Interactions with the
Arp2/3 complex are critically dependent upon the last three
residues in the C terminus of the WA domain, which are readily lost
during preparation of this domain from glutathione-S-transferase
(GST)-fusion proteins. Neither profilin nor actophorin competes
for binding to the Arp2/3 complex.
A crucial finding is that actin filaments enhance binding of the
Arp2/3 complex to the WA domain. As we detected no binding of
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articles
WASP-WA to actin filaments, this effect is presumably a result of
binding of the Arp2/3 complex to the side of actin filaments.
The Arp2/3 complex enhances actin nucleation, the rate-limiting
step in polymerization. However, the Arp2/3 complex alone is inactive and requires activation by ActA or WASP/Scar proteins. Here we
have provided several insights into this activation mechanism.
The WA domain activates actin-filament nucleation by the
Arp2/3 complex, whereas other domains (WH-2, C, CA and A) or
mixtures of these separate domains have minimal or no activity in
spite of their residual affinity for either actin or the Arp2/3 complex. The simplest hypothesis to explain activation is that the WA
domain stabilizes binding of an actin monomer to the Arp2/3 complex (Fig. 1b). A new barbed end may simply grow from a trimeric
nucleating assembly consisting of an actin monomer, Arp2 and
Arp3. Filaments may promote nucleation by enhancing binding of
the WA domain to the Arp2/3 complex, prolonging the lifetime of
the actin–WASP-WA–Arp2/3 complex to favour the formation of a
productive nucleus.
Much is explained by the hypothesis that the WA domain serves as
an adaptor to bring together an actin monomer and the Arp2/3 complex, but the lack of correlation between the affinities of mutant
WASP-WA constructs for actin and the Arp2/3 complex and their
ability to activate actin nucleation by the Arp2/3 complex shows that
nucleation must involve further activation steps that occur subsequent to binding. Although neither of the WAS mutations K476E or
R477K alters affinity for actin or for the Arp2/3 complex, WASPWA(K476E) activates actin nucleation by the Arp2/3 complex better
than the wild-type, whereas WASP-WA(R477K) has reduced activity.
Similarly, our mutation N445K causes a fourfold increase in affinity
for actin, wild-type affinity for the Arp2/3 complex and slightly
enhanced ability to activate nucleation by the Arp2/3 complex, whereas our mutation K446T gives rise to a threefold reduction in affinity
for actin, wild-type affinity for the Arp2/3 complex and slightly
enhanced activation of the Arp2/3 complex. Rhodamine labelling of
WASP-429-WA shows that increased affinity for actin and the Arp2/3
complex reduces, rather than enhances, nucleation by the Arp2/3
complex. Even mutant WASP-WA(W500S), which has highly reduced
affinity for the Arp2/3 complex, can stimulate nucleation at high concentrations. Finally, it will be interesting to learn why dimeric
GST–WASP-WA and full-length WASP activate the Arp2/3 complex
100 times more effectively than the monomeric WA domain22.
All of these observations indicate a delicate balance between
affinity and dynamics when WASP activates nucleation by the
Arp2/3 complex. As WASP and ActA immobilized on beads can
drive Arp2/3-complex-dependent assembly of an actin-filament
halo14,35, these activators must dissociate from the Arp2/3 complex
soon after initiating nucleation. The rapid dissociation of the WA
domain from both actin and the Arp2/3 complex observed here
account for these observations. Further information is still needed
to understand the activation mechanism and the physiological
defects caused by WAS mutations.
an additional N-terminal cysteine codon. Cys–WASP-WA was cloned into pGEX 2T (Amersham) and
expressed in BL21 (DE3) pLysS E. coli. Fusion protein was purified by glutathione–Sepharose affinity
chromatography (Amersham) according to the manufacturer’s instructions. WASP-WA peptide was
cleaved from GST by incubation for 2 h at 4 °C with 2.5 NIH units of bovine thrombin (Calbiochem)
per mg of immobilized GST–WASP-WA. Thrombin was inactivated with 1 mM phenylmethylsulphonyl fluoride (PMSF). Thrombin cleavage removes two or more C-terminal residues from a substantial
fraction of WASP-WA. These inactive degradation products are not resolved from full-length WASPWA by SDS–PAGE, but can be separated by MonoQ chromatography (Amersham) equilibrated with
TBSE (10 mM Tris–HCl pH 8.0, 100 mM NaCl and 1 mM EDTA) and eluted with a gradient of
100–400 mM NaCl. The –SH of unlabelled WASP-WA was reacted with a fivefold molar excess of Nethyl maleimide.
GST–WASP-WA constructs attached to glutathione beads were labelled by incubating 1 ml of a
50% resin suspension overnight at 4 °C in 1.6 mM tetramethylrhodamine-5′-maleimide (Molecular
Probes) in TBSE. The resin was washed extensively with TBSE. Rhodamine–WASP-WA was released by
cleavage with 15 U thrombin per mg of protein, and purified further as described for the unlabelled
protein. Electrospray mass spectrometry at the Scripps Research Institute gave Mr values of 8,648 for
rhodamine–WASP-429-WA, 8,820 for rhodamine–WASP-427-WA, 9,061 for rhodamine–WASP-424WA and 10,636 for rhodamine–WASP-406-WA, confirming that each purified peptide was intact and
100% labelled with a single dye.
Concentrations of unlabelled and labelled WASP-WA were determined by absorbance. The extinction coefficient of 44,656 M–1 cm–1 at 552 nm of rhodamine–WASP-WA was determined by Raleigh
interference and absorption in a Beckman XLI analytical ultracentrifuge. For unlabelled WASP-WA, an
extinction coefficient of 5,690 M–1 cm–1 at 280 nm was calculated by the same method.
Preparation of WASP-WA mutants.
An EcoRI site present next to the 443QLNK446 motif of WASP was used to generate mutations
Q443R, N445K and K446T by PCR using primers carrying point mutations and EcoRI sites. Amplified
fragments were cloned into pGEX 2T WASP-WA digested by EcoRI. The W500S mutant was engineered by amplification of WASP-WA using a reverse primer carrying the point mutation.
Wiskott–Aldrich-syndrome mutations K476E, R477K and D485N were generated by reverse PCR
mutagenesis38. Mutations were verified by DNA sequencing.
Fluorescence anisotropy.
Anisotropy measurements were made with an Alpha-scan spectrofluorimeter (Photon Technology
International, Santa Clara, California) with samples in KMEI buffer (10 mM imidazole pH 7.0, 50 mM
KCl, 1 mM EGTA and 1 mM MgCl2)25. Fixed concentrations of rhodamine–WASP-WA were mixed
with various concentrations of actin or of the Arp2/3 complex. Rhodamine was excited with polarized
light at 552 nm and the emitted light was detected at 574 nm through both horizontal and vertical
polarizers. Measurements were made at 1-s intervals for 20 s, and the average anisotropy was calculated
using PTI Felix software. Dissociation-equilibrium constants (Kd) were calculated by fitting equation
(1) to the ligand-concentration dependence of the anisotropy.
r = rf + (rb - rf) (Kd + [W] + [L]) –
(Kd + [W] + [L])2 – (4[W][L])
(1)
2
where rf is the anisotropy value of free rhodamine–WASP-WA, rb is the anisotropy of
rhodamine–WASP-WA bound to ligand, [W] is the concentration of rhodamine–WASP-WA, and [L] is
the concentration of ligand (actin or the Arp2/3 complex).
Competition experiments.
In experiments in which a non-fluorescent ligand competed with rhodamine–WASP-WA for binding
to actin or to the Arp2/3 complex, equation (2) was fitted to the dependence of anisotropy on the concentration of competitor.
r = rf + (rb - rf )/[(K d([C] + K 2)/K 2 [L]) + 1]
(2)
where Kd is the dissociation-equilibrium constant of labelled WASP-WA and actin or the Arp2/3 complex, [L] is the total concentration of either actin or the Arp2/3 complex, [C] is the concentration of
competing ligand, and K2 is the Kd of the competing ligand for actin or for the Arp2/3 complex.
Methods
Protein preparation.
Ca-ATP–actin was purified from an acetone powder of rabbit skeletal muscle and monomers were
purified by gel filtration on Sephacryl S-300 in G-buffer (2 mM Tris–HCl pH 8.0, 0.2 mM ATP,
0.1 mM CaCl2 and 0.5 mM dithiothreitol)36. Actin was labelled with pyrenyliodoacetamide (Molecular
Probes)32. Mg-ATP–actin was prepared by incubating Ca-ATP–actin with 0.1 mM MgCl 2 and 1.0 mM
EGTA for 2 min at room temperature immediately before each measurement. Recombinant humanplatelet profilin-I and actophorin were purified from Escherichia coli37. Thymosin-β4 was a gift from S.
Zigmond (Univ. Pennsylvania). The Arp2/3 complex was purified from bovine thymus8,22. Human
Scar1-WA (residues 495–559) was expressed in E. coli from the pMW vector. Cells were lysed by sonication and Scar1-WA in the soluble fraction was purified to homogeneity by gel filtration in 100 mM
KCl, 1 mM sodium azide, 0.5 mM EDTA and 10 mM Tris–HCl pH 8.0 and ion-exchange chromatography on diethylaminoethyl (DEAE)–cellulose with elution on a gradient of 100–500 mM KCl. Scar1WA has a predicted Mr of 7,646.3; mass spectroscopy gave a single peak corresponding to Mr 7,650.
Preparation and labelling of WASP-WA.
DNA corresponding to the C terminus of human WASP (residues 429–502) was amplified from a
cloned complementary DNA10 by polymerase chain reaction (PCR) with a forward primer containing
Dissociation kinetics.
The time course of dissociation of rhodamine–WASP-WA from actin or from the Arp2/3 complex was
determined by recording the reduction in anisotropy after mixing equilibrated samples of rhodamine–WASP-WA bound either to actin or to the Arp2/3 complex with a 100–200-fold molar excess
of Scar-WA25, using a KinTek stopped-flow mixer with a dead time of ~7 ms. The time course of
anisotropy change followed a single exponential.
Assay of actin-filament nucleation.
The time course of actin polymerization was used to assess the ability of WASP-WA constructs to activate nucleation by the Arp2/3 complex8. Reactions contained 4 µM Mg-ATP–actin monomers (5%
pyrene-labelled), 10 nM bovine thymus Arp2/3 complex and a range of WASP-WA concentrations all
in KMEI buffer. Polymerization was followed by fluorescence with excitation at 365 nm and emission
at 407 nm. At every time point the rate of polymerization is equal to k+(ends)(actin monomers), where
k+ is the association rate constant (10 µM–1 s–1). The concentration of ends at the maximum polymer-
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81
articles
ization rate was calculated. The products of these nucleation reactions were observed by fluorescence
microscopy3.
Isothermal titration calorimetry.
The heat produced during binding of Scar1-WA to actin monomers was measured with an Omega
titration calorimeter (MicroCal Inc., Northampton, Massachusetts) with a 1.4-ml reaction cell. Scar1WA was injected in 5-µl aliquots into an actin solution. To allow for accurate determination of the biochemical standard-state enthalpy change upon binding, we chose concentrations of reactants such that
the product (Ka × initial actin concentration × stoichiometry) was >> 1 (10–100 in our experiments).
Buffers and temperatures were as described in the text. Heats of dilution of Scar1-WA into actin saturated with WASP-WA were subtracted from each injection heat. Origin software from MicroCal was
used to determine peak areas and to analyse the data; the entropy change (∆S) was calculated using
∆G = ∆H – T∆S.
Circular-dichroism spectroscopy.
Circular-dichroism spectra of WASP-WA and Scar1-WA in 10 mM sodium phosphate buffer, pH 7.2,
were collected at 25 °C with an Aviv Model 62DS spectropolarimeter, using the manufacturer’s software. Spectra were deconvoluted using four methods 39–42.
RECEIVED 31 MAY 2000; REVISED 17 JULY 2000; ACCEPTED 11 SEPTEMBER 2000;
PUBLISHED 7 DECEMBER 2000.
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ACKNOWLEDGMENTS
We thank L. Blanchoin, K. Amann, W-L. Lee (analytical ultracentrifugation), S. C. Koerber (circulardichroism spectroscopy) and J. Meisenhelder of the Salk Institute for help with this work. This work
was supported by NIH Research Grant GM-26338 (to T.D.P.), an NIH Postdoctoral Fellowship (to
H.N.H.), a postdoctoral fellowship from Association pour la Recherche sur le Cancer (ARC) and the
Philippe Foundation (to J.B.M.), and a gift to the laboratory from S. Hammerslag.
Correspondence and requests for materials should be addressed to T.D.P.
NATURE CELL BIOLOGY VOL 3 JANUARY 2001 http://cellbio.nature.com
© 2000 Macmillan Magazines Ltd
supplementary information
a
b
c
d
e
M r (K)
43
Supernatants
29
18
14
M r (K)
43
Pellets
29
18
14
1
2
3
4
5
6
7
8
Figure S1 Mutations in WASP-WA do not affect actin-filament branching or
interaction with WASP-GBD. a–d, Fluorescence micrographs of actin filaments
polymerized in 50 mM KCl, 1 mM EGTA, 1 mM MgCl2, 10 mM imidazol pH 7.0, 4
µM actin, 20 nM Arp2/3 complex and 4 µM rhodamine–phalloidin. Samples were
diluted 500-fold into stabilization buffer and applied to nitrocellulose-coated coverslips. a, No WASP-WA. b, 2 µM WASP-429-WA. c, 4 µM WASP-WA(W500S). d, 2.6
µM WASP-WA(R477K). Scale bar represents 10 µm. e, Binding of WA-domain constructs to WASP-GBD. WASP(152–309) (2 µM) and glutathione–Sepharose beads
bound to GST alone (lane 1), GST–WASP-429-WA (lane 2), GST–N-WASP-WA (lane
3), GST–Scar1-WA (lane 4), GST–WASP-WA(K476E) (lane 5), GST–WASP-WA(R477K)
(lane 6) or GST–WASP-WA(D485N) (lane 7) were incubated for 5 min at 23 °C and
then centrifuged at 5,000g for 5 min. Equal volumes of pellets and supernatants
were analysed by Coomassie staining after SDS–PAGE. Arrow indicates
WASP(152–309); higher bands are GST fusions. Lane 8 shows 2 µM
WASP(152–309) for comparison.
Table S1 Titration calorimetry
Conditions
50 mM KCl, 10 mM imidazole
Stoichiometry
Kd
(µM)
∆G
(kcal mol–1)
∆H
(kcal mol–1)
∆S
(cal mol–1 K–1)
1.0
0.2
–9.1
–10.1
–3.4
1.1
2.2
–7.7
–9.1
–4.5
pH 7.0, 1 mM EGTA, 1 mM
MgCl2, 0.2 mM ATP, 0.5 mM
dithiothreitol, 0.02% NaN3 and
50 µM Latrunculin B
2 mM imidazole pH 7.0, 0.2
mM ATP, 0.5 mM dithiothreitol and 0.02%
NaN3
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