Actin-bound structures of Wiskott–Aldrich syndrome
protein (WASP)-homology domain 2 and the
implications for filament assembly
David Chereau, Frederic Kerff, Philip Graceffa, Zenon Grabarek, Knut Langsetmo, and Roberto Dominguez*
Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472
Edited by Thomas D. Pollard, Yale University, New Haven, CT, and approved September 28, 2005 (received for review August 12, 2005)
Wiskott–Aldrich syndrome protein (WASP)-homology domain 2
(WH2) is a small and widespread actin-binding motif. In the WASP
family, WH2 plays a role in filament nucleation by Arp2?3 complex.
Here we describe the crystal structures of complexes of actin with
the WH2 domains of WASP, WASP-family verprolin homologous
the actin monomer-sequestering thymosin ? domain (T?). We
show that both domains inhibit nucleotide exchange by targeting
for many unrelated actin-binding proteins. Importantly, WH2 is
significantly shorter than T? but binds actin with ?10-fold higher
affinity. WH2 lacks a C-terminal extension that in T?4 becomes
involved in monomer sequestration by interfering with intersub-
unit contacts in F-actin. Owing to their shorter length, WH2
domains connected in tandem by short linkers can coexist with
intersubunit contacts in F-actin and are proposed to function in
filament nucleation by lining up actin subunits along a filament
strand. The WH2-central region of WASP-family proteins is pro-
posed to function in an analogous way by forming a special class
of tandem repeats whose function is to line up actin and Arp2
for how profilin-binding Pro-rich sequences positioned N-terminal
to WH2 could feed actin monomers directly to WH2, thereby
playing a role in filament elongation.
x-ray crystallography ? isothermal titration calorimetry ? nucleotide
cell shape and polarity (1). In the cell, a vast number of
actin-binding proteins (ABPs) direct the location, rate, and
timing for actin assembly into different structures, such as
filopodia, lamellipodia, stress fibers, and focal adhesions. ABPs
are commonly multidomain proteins, containing signaling do-
mains and structurally conserved actin-binding motifs. One of
the most abundant actin-binding motifs is Wiskott–Aldrich
syndrome protein (WASP)-homology domain 2 (WH2) (2). The
hematopoietic-specific protein, WASP, and its ubiquitously ex-
pressed ortholog N-WASP form part of a family that also
includes the three WASP-family verprolin homologous protein
(WAVE?SCAR) isoforms: WAVE1, WAVE2, and WAVE3 (1,
3). Members of this family activate Arp2?3-dependent actin
nucleation and branching in response to signals mediated by
Rho-family GTPases. Although the domain structure of these
proteins varies, reflecting different modes of regulation, they all
share a common C-terminal WH2 central-acidic region (CA
region) (Fig. 1A), which constitutes the smallest fragment nec-
of the WASP-interacting protein (WIP) family, which form
complexes with WASP?N-WASP and modulate their functions
in vivo (5, 6). Members of this family include WIP, CR16, and
WIRE (or WICH) in mammals and verprolin in yeast.
he actin cytoskeleton plays an essential role in many cellular
functions, including intracellular transport and the control of
It has been proposed, based on sequence analysis, that WH2
forms part of an extended family with the thymosin ? domain
(T?) (7). However, this view is controversial, in part because of
the different biological functions and low sequence similarity of
WH2 and T? (8). The actin-bound structures of the N-terminal
half of ciboulot domain 1 (9) and that of a hybrid protein
consisting of gelsolin domain 1 and the C-terminal half of T?4
(10) have been reported. These structures have been combined
into a model of T?4–actin (10), and, although both T?4 and
ciboulot belong in the T? family, their structures have been
described as representatives of WH2 (9, 10).
Here we report the crystal structures of complexes of actin
with the WH2 domains of WASP, WAVE2, and WIP, in parallel
with a biochemical characterization of WH2 and T?. Important
structural and biochemical differences set apart WH2 and T? in
accordance with their specialized cellular functions. The struc-
tures of WH2–actin shed light on the molecular principles
supporting the role of WH2 in filament nucleation and
Materials and Methods
Proteins and Peptides. Actin was prepared and labeled with
acrylodan as described in ref. 11. Ultrapure-grade bovine pan-
creatic DNase I was purchased from BioWorld (Dublin, OH).
Peptides corresponding to WASP430–458, WAVE2433–464,
WAVE2450–464, WIP29–60, WIP29–46, WIP46–63, MIM724–755,
T?42–44, T?42–33, T?418–44, ciboulot10–43, ciboulot49–81, and ci-
boulot87–119were synthesized on an ABI431 peptide synthesizer
and then purified by HPLC. The concentrations of the peptides
were determined by amino acid analysis (Dana–Farber Cancer
Crystallization, Data Collection, and Structure Determination. WH2–
actin–DNase I complexes were prepared at a 1:1:1.5 molar ratio,
dialyzed against G-buffer (2 mM Tris, pH 7.5?0.2 mM CaCl2?0.2
mM ATP?1 mM NaN3), and concentrated to ?10 mg?ml using
a Centricon device (Millipore). The complexes were crystallized
hanging drops consisted of a 1:1 (vol?vol) mixture of protein
solution and a well solution containing 13–14% polyethylene
glycol 3350, 50 mM Na cacodylate (pH 6.8–7.2), and 100 mM Na
formate. The crystals were flash-frozen in propane, with 25%
glycerol as a cryoprotectant. X-ray data sets were collected at
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: WASP, Wiskott–Aldrich syndrome protein; WH2, WASP homology domain
2; WAVE, WASP-family verprolin homologous protein; WIP, WASP-interacting protein;
ABP, actin-binding protein; T?, thymosin ? domain; ITC, isothermal titration calorimetry;
Data deposition: The atomic coordinates reported in this paper were deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 2A40, 2A41, 2A42, and 2A3Z).
*To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
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no. 46 www.pnas.org?cgi?doi?10.1073?pnas.0507021102
to the number of WH2 repeats. This finding led to the suggestion
that the WH2 domains of spire line up actin subunits along a
filament strand of the actin double helix, thereby generating
nuclei for actin assembly (26). The Pro-rich sequences that
(27). Profilin is the major actin monomer carrier in the cell (1).
stimulates actin filament elongation by VASP (28) and WASP?
WAVE (29–31). The role of profilin in elongation appears to be
common among cytoskeletal proteins. Formins, for instance,
function in a similar way, promoting elongation in a profilin-
dependent manner by combining an actin binding FH2 domain
C-terminal to a Pro-rich FH1 domain, which mediates profilin
The WH2–actin structures show how WH2 could play a role
in both nucleation and elongation (Fig. 4A). The WH2 domains
of spire, similar to those in WASP?WAVE, are of the short kind
(?17 aa), and are connected by short linkers, which has two
WH2 domains does not interfere with intersubunit contacts in
F-actin and can thereby coexist with the actin filament, as
illustrated by a superimposition of a WH2–actin structure onto
two consecutive subunits of Holmes’ filament model (32) (Fig.
along the same filament strand because the size of the linkers
cannot support connections between actin subunits on opposite
filaments strands. The actin subunits brought together by this
type of interaction can form the nucleus for a new filament, as
observed in spire (26).
As noticed before (9, 10), the C region of WASP?WAVE
proteins bears similarity with WH2 (Fig. 1B). Therefore, WH2
C region could represent a specialized form of tandem repeat
involved in Arp2?3 nucleation. Like WH2, the N-terminal
portion of the C region forms an amphiphilic ?-helix (33).
Mutation of Leu-471 on the hydrophobic side of this ?-helix
impairs Arp2?3-mediated nucleation. As demonstrated here, the
?-helix of WH2 binds in the cleft between actin subdomains 1
and 3, accounting for most of the actin-binding affinity and
nucleotide exchange inhibition of WH2. This cleft in actin
constitutes a ‘‘hot spot’’ for numerous ABPs (14). Although
Arp2 is partially disordered in the structure of Arp2?3 complex
(34), a model based on the structure of actin suggests that this
cleft is conserved in Arp2, whereas a C-terminal extension of the
polypeptide chain partially occupies the cleft in Arp3. Therefore,
by analogy with tandem WH2 domains, WH2 C region is
proposed to line up an actin subunit and Arp2 along a strand
of the daughter filament branch during Arp2?3 nucleation
The Pro-rich regions preceding WH2 domains may facilitate
filament elongation in two possible ways: by increasing the local
concentration of profilin–actin, consistent with the stimulation
of assembly by profilin-WASP?WAVE (29–31) and?or by me-
diating actin monomer transfer directly to WH2 via profilin. The
latter corresponds to the ‘‘loading’’ step in the so-called actoc-
lampin motor model (35), in which an actin-binding domain (or
clamp) translocates processively at the growing filament end in
response to ATP hydrolysis by actin. We propose that in proteins
such as VASP (Fig. 1B) WH2 forms such a clamp. Because WH2
has higher affinity for ATP–actin than ADP–actin (Table 3),
ATP hydrolysis by actin could provide the energy necessary for
the processive stepping of WH2-based motors. The structures of
WH2–actin, together with those of profilin–actin (19) and
profilin-polyPro (36), suggest that profilin bound to the consen-
sus Pro-rich sequence immediately N-terminal to WH2 could
deliver its actin directly to WH2, contributing to barbed-end
filament elongation (Figs. 1B and 4C). A slight overlap between
the actin-binding sites of profilin and WH2 (Fig. 2H) and?or
allosteric effects may then release profilin from the growing
This work was supported by National Institutes of Health Grant
GM073791. Use of the Advanced Photon Source was supported by the
U.S. Department of Energy, Basic Energy Sciences, Office of Science,
under Contract W-31-109-Eng-38. Use of Industrial Macromolecular
Crystallography Association Collaborative Access Team Beamline
17-ID was supported through a contract with the University of Chicago.
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