Structure 14, 159 166, January 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.str.2005.10.005
Structure of the Forkhead Domain
of FOXP2 Bound to DNA
James C. Stroud,1,4,5 Yongqing Wu,1,4,5 1999), and thyroid agenesis with cleft palate and choa-
Darren L. Bates,1,4 Aidong Han,1,4 Katja Nowick,2 nal atresia (FOXE1) (Clifton-Bligh et al., 1998). Often,
* *
Svante Paabo,2 Harry Tong,3, and Lin Chen1,4, these mutations are within the well-conserved forkhead
1
Department of Chemistry and Biochemistry domain (Carlsson and Mahlapuu, 2002), demonstrating
University of Colorado at Boulder the importance of DNA recognition and binding to the
Boulder, Colorado 80309 function of FOX proteins.
2
Max Planck Institute for Evolutionary Anthropology FOXP (FOXP1 4) is a newly defined subfamily of the
D-04103 Leipzig FOX transcription factors that contains several recogniz-
Germany able sequence motifs, including a glutamine-rich region,
3
Australian Synchrotron Research Program/ a zinc finger, a leucine zipper, and a highly divergent
Consortium for Advanced Radiation Sources forkhead domain (Lai et al., 2001; Li and Tucker, 1993;
Argonne National Laboratory Shu et al., 2001). As seen in several other FOX proteins
Building 434-B linked to human developmental disorders (Lehmann
9700 South Cass Avenue et al., 2003), the majority of disease-causing mutations
Argonne, Illinois 60439 in FOXP2 and FOXP3 occur in the forkhead domain. For
instance, an arginine-to-histidine missense mutation
(R553H) in the FOXP2 forkhead domain has been linked
Summary to a severe speech and language disorder (Lai et al.,
2001). A deletion of the forkhead domain arising from a
FOXP (FOXP1 4) is a newly defined subfamily of the frame-shift mutation in the FOXP3 protein in mouse is
forkhead box (FOX) transcription factors. A mutation linked to the autoimmune disorder scurfy (Brunkow
in the FOXP2 forkhead domain cosegregates with a se- et al., 2001; Schubert et al., 2001). A similar congenital
vere speech disorder, whereas several mutations in disease in human is known as IPEX (immune dysregula-
the FOXP3 forkhead domain are linked to the IPEX syn- tion, polyendocrinopathy, enteropathy, X-linked syn-
drome in human and a similar autoimmune phenotype drome) (Bennett et al., 2001; Wildin et al., 2001). Afflicted
in mice. Here we report a 1.9 Å crystal structure of the individuals display a variety of symptoms that include
forkhead domain of human FOXP2 bound to DNA. This anemia, insulin-dependent diabetes, chronic diarrhea,
structure allows us to revise the previously proposed and dermatitis (Levy-Lahad and Wildin, 2001). The sim-
DNA recognition mechanism and provide a unifying ilarities between human IPEX and mouse scurfy pheno-
model of DNA binding for the FOX family of proteins. types are reflected by the fact that several mutations in
Our studies also reveal that the FOXP2 forkhead do- the forkhead domain of the human FOXP3 gene have
main can form a domain-swapped dimer, made possi- been linked to IPEX (Bennett et al., 2001; Wildin et al.,
ble by a strategic substitution of a highly conserved 2001).
proline in conventional FOX proteins with alanine in To address the mechanisms by which these disease-
the P subfamily. Disease-causing mutations in FOXP2 related mutations disturb FOXP function, we have deter-
and FOXP3 map either to the DNA binding surface mined the structure of the human FOXP2 forkhead
or the domain-swapping dimer interface, functionally domain bound to DNA containing a FOXP binding site
corroborating the crystal structure. (Schubert et al., 2001; Wang et al., 2003) (Table 1). Our
results show that disease-causing mutations in the
FOXP family map to the DNA binding interface and to
Introduction
a dimer interface formed by domain swapping. Domain
swapping can be disrupted by replacing an alanine con-
Forkhead box (FOX)-containing transcription factors are
served in the FOXP family with a proline that is highly
unified by sequence similarity within an approximately
conserved in other FOX families. These results suggest
90 amino acid winged-helix DNA binding domain from
that domain swapping is a unique structural feature of
which the FOX family derives its name (Mazet et al.,
the FOXP forkhead domain and thus may be functionally
2003). The diverse roles of FOX (human protein names
relevant. Additionally, the high resolution of the data
are used throughout) family members in development
allows us to reinterpret earlier, lower resolution studies
are underscored by the fact that mutations in several
and propose a general model of DNA recognition by
members of the family are linked to congenital defects,
forkhead-containing proteins.
including familial glaucoma and Axenfeld-Rieger anom-
alies (FOXC1) (Lehmann et al., 2000; Mears et al., 1998;
Results and Discussion
Mirzayans et al., 2000; Nishimura et al., 1998, 2001), lym-
phedema (FOXC2) (Fang et al., 2000; Finegold et al.,
Overall Structure
2001), T cell immunodeficiency (FOXN1) (Frank et al.,
The asymmetric unit (ASU) contains six copies of the
FOXP2 forkhead domain and two double-stranded seg-
ments of DNA (Figure 1A). Although all six copies of
*Correspondence: lin.chen@colorado.edu (L.C.); tong@cars.uchicago.
FOXP2 are identical in sequence, two copies exist in a
edu (H.T.)
4
monomeric form (Figure 1A, labeled 1 and 2) and the four
Lab address: http://keres.colorado.edu/
5
These authors contributed equally to this work. other copies exhibit domain swapping (described in
Structure
160
extensively with both helix H3 and the sugar-phosphate
Table 1. Statistics of Crystallographic Analysis
backbone, thereby wedging helix H3 deep into the major
Data Collection
groove and stabilizing the protein-DNA complex (Fig-
ure 2A). In the periphery of the FOXP2/DNA interface,
Resolution (Å) 40.0 1.90
Rsym (%)a 0.055
residues from the N and C termini (Arg504, Thr508,
Completeness (%)b 99.7 (99.1)
Arg583, and Arg584), including the main chain amide
I/sb 28.46 (4.8)
of Tyr509 at the N-terminal end of helix H1 and residues
Refinement
from S2 (e.g., Arg564), make hydrogen bonds, van der
Waals contacts, and electrostatic interactions with the
Resolution (Å) 40.0 1.90
R factorb,c 0.217 (0.243) DNA backbone, providing further stability to the FOXP2/
Rfreeb,c 0.235 (0.278)
DNA complex.
Rms deviations
Based on the major groove contacts, the DNA binding
Bond lengths (Å) 0.007
site of FOXP2 can be defined as 50-CAAATT-30(the core
Bond angles (º) 1.1
binding sequence is in bold) (Figure 2B), which is similar
Average B factor (Å2) 37.6
to that derived from in vitro selection (50-A[C/T]AAATA-
a
Rsym = SjI 2 j/S I, where I is the observed intensity and is the
30) (Wang et al., 2003). DNA binding by FOXP2 shares
statistically weighted average intensity of multiple observations of
a similar global structure with the FOXA3/DNA complex
symmetry-related reflections.
b
(Clark et al., 1993). Surprisingly, the binding site deter-
Numbers in parentheses are for the outer shell.
c
R factor = SkFoj2jFck/SjFoj, wherejFojandjFcjare observed and mined for FOXA3 in the previous crystallographic study
calculated structure factor amplitudes, respectively. Rfree is calcu- was 50-TAAGTCAACC-30 (underlined), significantly dif-
lated for a randomly chosen 9.1% of reflections.
ferent from that seen here for FOXP2. The DNA binding
mechanism of FOXA3 derived from the early study was
detail below; Figure 1A, labeled 3 6). The two FOXP2
also significantly different from that described for
monomers bind intimately to equivalent sites on the two
FOXP2 above. However, upon careful examination, we
segments of DNA (described below), whereas the two
found that conserved residues on helix H3 of FOXA3
swapped dimers loosely associate with DNA. The DNA- (corresponding to Arg553, Asn550, His554, and Ser557
bound monomeric form folds into the canonical winged- in FOXP2) bind a DNA region (bold) in 50-TAAGTCA
helix motif characteristic of the FOX family (Clark et al.,
ACC-30 similarly to their counterparts in FOXP2. Nota-
1993) (Figure 1B). Its core is comprised of three stacking
bly, these residues are highly conserved among all
a helices (H1, H2, and H3) capped at one end by a three- known members of the FOX family (Figure 1C). We pro-
stranded antiparallel b sheet (S1, S2, and S3). The turn
pose that this region is a cryptic FOX binding site in the
between H2 and H3 contains a 310 helix (H4) as seen in
FOXA3/DNA complex (Clark et al., 1993). The revised
other FOX proteins (Clark et al., 1993; Jin et al., 1999;
interpretation of the DNA binding mechanism is not a re-
Liu et al., 2002; Weigelt et al., 2001).
sult of the different DNA sequences used in the FOXA3/
Between strands S2 and S3, conventional FOX pro- DNA complex and the FOXP2/DNA complex, as con-
teins contain a 5 7 amino acid insert, called wing 1.
served DNA binding residues of FOXA3 and FOXP2 en-
However, in FOXP2 this insert is truncated, resulting in
gage in similar DNA binding interactions in the two com-
a simple type I turn that joins strands S2 and S3 (Fig- plexes. Thus, based on the common features of protein/
ure 1C). The C-terminal region also distinguishes the
DNA interactions in the FOXA3/DNA complex and the
FOXP subfamily from most other FOX proteins. In FOXA3
present structure, we are able to redefine the FOX bind-
this region forms an extended loop, called wing 2 (W2),
ing sequence (50-CAAATT-30) at the structural level,
that contacts DNA extensively (Clark et al., 1993). The
which is consistent with the footprinting of FOXA3 and
corresponding region in FOXP2 forms a helix (H5) that
biochemical data on the binding site of a number of FOX
runs atop H1 and terminates at the DNA phosphate
proteins, including FOXK1 (50-TAAACA-30), FOXC2 (50-
backbone (Figure 1B). A similar helix H5 is also observed
GTAAACA-30), and FOXD1 (50-AAAATAAC-30) (Costa
in the NMR structures of FOXD1 and FOXK1a (ILF-1), but
et al., 1989; Jin et al., 1999; Liu et al., 2002; Nirula
the sequences and trajectories of these helices are nota- et al., 1997; van Dongen et al., 2000). A major difference
bly different from that of FOXP2 (Jin et al., 1999; Liu
in DNA binding between FOXP2 and FOXA3 is at the pe-
et al., 2002). The heightened variability of the W1 and
ripheral protein/DNA interface, where FOXA3 uses two
W2 regions relative to the rest of the forkhead domain
loops (W1 and W2) to bind the DNA backbone and minor
across all FOX subfamilies suggests that the wings may
groove extensively. The corresponding loops in FOXP2
have specialized functions within each subfamily.
are much shorter and make limited DNA contacts (Clark
et al., 1993). Consistent with these structural observa-
DNA Recognition tions, the forkhead domain of FOXP2 binds DNA with
DNA recognition by FOXP2 is mediated predominantly a lower affinity than that of FOXA3 (Clark et al., 1993;
by helix H3 (Figure 2). Asn550 forms bidentate hydrogen Li et al., 2004).
bonds with Ade10, whereas His554 and Arg553 form di- Compared with most sequence-specific transcription
rect or water-mediated hydrogen bonds with Thy100and factors, an unusual feature of DNA binding by FOXP2 is
Thy110, respectively. The main chain and side chain its extensive utilization of van der Waals contacts and a
atoms of Arg553, His554, Ser557, and Leu558 also make relatively small number of hydrogen bonds to bases in
extensive van der Waals contacts to Cyt8, Gua80, Thy90, the major groove. This shape recognition may allow
Thy100, Thy110, Ade120, and Ade130. A number of aro- FOXP2 to bind a broad range of sequences in different
matic or hydrophobic residues from helix H1 (Tyr509), promoter contexts as long as the DNA maintains the
H2 (Leu527 and Tyr531), and strand S3 (Trp573) interact few hydrogen bond determinants in the core region of
Structure of FOXP2 Bound to DNA
161
Figure 1. Overall Structure
(A) The asymmetric unit. FOXP2 molecules
are shown as ribbon drawings. Molecules
within the swapped dimers are both orange
(3 and 5) and cyan (4 and 6). Monomers (1
and 2) are orange. The DNA phosphate back-
bone is shown as a coil in magenta.
(B) Ribbon drawing of FOXP2 in the mono-
meric form bound to DNA. The sequence of
the region of DNA pictured is below the com-
plex. DNA is shown as wire frame.
(C) Sequence alignment. FOXP proteins are
separated from other FOX proteins by a
dashed line. Differences in secondary struc-
ture in the first half of the protein (residues
503 544) between the monomer (Mono, or-
ange) and the dimer (Swap, cyan) are shown
below the sequence. The second half (resi-
dues 545 584) has the same secondary
structure in the monomer (shown in orange)
and dimer (not shown). Residues that require
significant backbone changes between the
monomeric and swapped forms are indicated
as a cyan dash superimposed on the mono-
meric secondary structure representation.
Residues involved in DNA binding (shaded
in magenta) and intermolecular interactions
in the swapped dimer (cyan circles above
the sequence) are highlighted. The arginine
(R553) linked to speech disorder is indicated
by a green filled box. Residues homologous
to those of FOXP3 linked to autoimmune dis-
eases are indicated by yellow filled boxes.
The alanine (A539) found to be critical for
swapping is shown with shaded background.
the binding site and has shape complementary to the forkhead domain binds its cognate site in two indepen-
DNA binding surface of FOXP2. Because DNA-contact- dent complexes of the crystal asymmetric unit. Second,
ing residues on H3 are almost absolutely conserved (Fig- the detailed binding interactions observed at the FOXP2/
ure 1C), this DNA binding behavior is likely common to all DNA interface are conserved in the FOXA3/DNA com-
FOX proteins, which do not recognize a single consensus plex. Finally, the DNA binding mechanism derived from
sequence but rather a degenerate pattern: 50-RYMAAYA-30 the FOXP2/DNA complex is consistent with biochemical
(R=AorG; Y=CorT; M=AorC) (Carlsson and Mahlapuu, data (see above). However, given the short recognition
2002). Although there is evidence that a leucine zipper sequence and relatively weak DNA binding affinity of
motif preceding the forkhead domain may be required the forkhead domain of FOXP (Li et al., 2004), it is likely
for high-affinity DNA binding by FOXP proteins (Li that specific DNA binding by FOXP proteins in vivo will
et al., 2004), our studies here suggest that the isolated be facilitated by protein/protein interactions in higher
FOXP forkhead domain is capable of specific DNA bind- order transcription factor complexes (Bettelli et al.,
ing based on a number of observations. First, the FOXP2 2005; Li et al., 2004).
Structure
162
Figure 3. Structure of Domain Swapping
(A) Top view of domain swapping in FOXP2. The two FOXP2 fork-
head domains are represented as orange (labeled) and cyan. Bottom
panel: two monomers have been placed in positions to illustrate the
rearrangements required for swapping.
(B) Side view of domain swapping. This view is rotated 90º around
the horizontal axis relative to (A).
(C) Stereodiagram of electron density around the core of the swap-
ped dimer interface.
(D) A number of hydrophobic residues exposed on the surface of the
monomeric FOXP2 bound to DNA. These residues include Pro506,
Phe507, Phe538, and Trp533. These residues become buried in the
domain-swapped dimer.
shows part of this interaction network around the 2-fold
axis of the swap. Here, Phe541 stacks face to face with
its pseudosymmetry mate, Phe538 packs face to edge
against Phe541 of its dimer partner, and Tyr540 stacks
edge to face against Phe541 of the same FOXP2 copy.
The swapped dimer buries several hydrophobic resi-
dues that are exposed in the DNA-bound monomeric
Figure 2. DNA Recognition species, including Pro506, Phe507, Trp533, and Phe538.
However, these residues are highly conserved in mono-
(A) Detailed interactions between the forkhead domain of human
FOXP2 (orange) and its cognate DNA site (magenta). The DNA and
meric FOX proteins (Clark et al., 1993) (Figure 3D). Thus,
protein residues are drawn as a stick model.
burial of exposed hydrophobic residues in FOXP2 must
(B) Schematic of interactions between FOXP2 and DNA. DNA is rep-
be supplemented by other factors that contribute to its
resented as a ladder with bases as ovals and labeled according to
propensity for domain swapping.
the text (the core sequence is highlighted by thick lines). The back-
Domain swapping in FOXP2 is a result of the exten-
bone phosphates are represented as circles with the letter P inside.
sion of helix H2 through the turn connecting H2 to H3
Hydrogen bonding interactions are solid arrows while van der Waals
interactions are dashed arrows. Secondary structure elements of
(Figures 3A and 4A), which creates a single straight 15
FOXP2 are boxed and labeled. Highly conserved residues that con-
amino acid a helix in place of the shorter helices of H2
tribute to DNA specificity in the FOXP2/DNA and FOXA3/DNA com-
and H4. This region, corresponding to residues 538
plexes are highlighted in red. A water molecule is represented as
541 (FAYF) in FOXP2, is highly conserved in all FOX pro-
a circle with a W inside.
teins, except for residue Ala539. In classical FOX pro-
teins, this position is occupied by a proline (Figure 1C),
Domain Swapping which most likely prevents the merging of helices H2
A striking structural feature of FOXP2 is its propensity to and H4 and therefore precludes domain swapping. This
form a domain-swapped dimer wherein two monomers proline is strategically replaced by an alanine residue in
of FOXP2 exchange helix H3, and strands S2 and S3 all FOXP members, suggesting that domain swapping is
(Figure 3A). The swapping buries an additional 804 Å2 a common feature in the FOXP family. Thus, contrary to
of solvent-accessible surface and creates a semicircular many cases of 3D domain swapping observed under
arch with a pseudo 2-fold symmetry (Figure 3B). Helices nonphysiological conditions or with artificially mutated
H2, H4, and H3 form the convex surface of the arch, proteins, it seems that domain swapping is an adaptive
while helices H1 and H5 form the concave surface. An structural feature of the P branch of FOX proteins. Con-
elaborate interaction network of aromatic residues sistent with this hypothesis, we have shown that the
spans the core of the swapped dimer. These residues in- forkhead domain of FOXP2 exists as both a monomer
clude Phe507, Tyr509, Tyr531, Trp533, Phe534, Phe538, and dimer in solution with a slow exchange rate (Fig-
Tyr540, Phe541, and Trp548 from both dimers. Figure 3C ure 4B) (see Experimental Procedures for further details).
Structure of FOXP2 Bound to DNA
163
Figure 4. Biochemical Analysis and Functional Implication of Do-
main Swapping
(A) Superposition of the domain-swapped FOXP2 dimer (cyan) and
the monomer (yellow) showing the region (blue) that undergoes sig-
nificant backbone conformational changes. This region corre-
sponds to residues 536 548 in human FOXP2, indicated by a dashed
line in Figure 1C.
(B) Multiangle light scattering (MALS) analysis of the wild-type hu-
man FOXP2 (residues 503 584). The profile shows two discrete
peaks corresponding to monomer (13.8 KD) and dimer (26.2 KD).
Blue lines: refractive index signal profile: MALS measurement of
mass at that point of the elution. The center of the peak gives the
greatest signal-to-noise for the measurement of mass.
(C) MALS analysis of the Ala539Pro mutant of human FOXP2 (resi-
dues 503 584) showing a single monodispersive peak correspond-
ing to monomer (14.1 KD).
(D) Electrostatic surface potential of the domain-swapped FOXP2 di-
mer (left). The DNA binding helix (H3) of one monomer in the domain-
swapped dimer inserts into the DNA major groove in a similar manner
to that seen in the monomer/DNA complex (shown on the right for
comparison), although its interaction with DNA is loose due to the
noncognate DNA sequence (not shown). The other monomer can
presumably bind a separate DNA substrate (Figure 5A) and the rela-
tively positive surface potential (blue) on top of the arch may facilitate
the binding of two strands of DNA (Figure 5B). Figure 5. A Specialized Function of FOXP Proteins May Be to Pro-
mote the Assembly of Higher Order Protein/DNA Complexes
(A) A model of the domain-swapped FOXP dimer (cyan and orange)
By contrast, the Ala539Pro mutant of FOXP2 exists
bound to two separated DNA sites (top view).
exclusively as a monomer in solution (Figure 4C). The
(B) A side view of the model. The leucine zipper (LZ) preceding the
mechanism by which this single amino acid change pre- forkhead domain, which may facilitate the formation of the domain-
swapped dimer, is also shown as a cylinder. In this view, the back-
vents swapping in classical FOX proteins appears to
bone of the two strands of DNA are closer on the top of the arch-
arise from proline s extraordinary propensity to disrupt
shaped dimer, where the protein has a relatively positive surface
a helices (Pace and Scholtz, 1998). Fortuitously, we
potential (see Figure 4D).
have found that the optimal molar ratio of protein to
(C) Proposed roles of the FOXP dimer in DNA looping (left) and inter-
DNA is 3:1 for crystallizing the FOXP2/DNA complex, al-
chromosomal interaction (right).
lowing us to observe both the monomer- and domain-
swapped dimer in the crystal. librium by the FOXP2 forkhead domain remain to be in-
The N termini of the FOXP2 forkhead domain in the vestigated. The two H3 helices in the swapped FOXP2
swapped dimer are close to each other (Figure 3B). In- dimer are separated sufficiently to allow both copies of
terestingly, FOXP proteins contain a highly conserved FOXP2 to bind DNA simultaneously. However, because
zinc finger/leucine zipper motif about 50 residues N- the two DNA binding surfaces are connected by a rigid
terminal to the forkhead domain. This motif has been protein domain characterized by extensive aromatic
shown to mediate dimerization of FOXP proteins (Li interactions (see above), the DNA binding sites of the
et al., 2004; Wang et al., 2003). In the full-length protein, domain-swapped FOXP2 dimer would need to be well-
this zinc finger/leucine zipper may cooperate with the separated from each other or from separate DNA strands
forkhead domain to facilitate the formation of domain- (Figure 5). Based on this structural feature, we propose
swapped dimers by FOXP proteins under physiological that a unique function of the FOXP family of proteins
concentrations. However, we cannot rule out the possi- is to loop DNA and/or mediate interchromosomal asso-
bility that the FOXP2 forkhead domain may also act as ciations. Consistent with this proposed role, the con-
a monomer to bind DNA in vivo. The thermodynamics vex surface is enriched in basic residues and has an
and functional implication of this monomer/dimer equi- overall positive electrostatic surface potential, which
Structure
164
may reduce phosphate backbone repulsion when two
double-stranded DNAs are brought together (Figure 4D).
Disease-Related Mutations
FOXP2 and FOXP3 were discovered as the targets of ge-
netic mutations in a human speech disorder (Lai et al.,
2001) and the autoimmune disease IPEX (Bennett et al.,
2001; Brunkow et al., 2001; Wildin et al., 2001), respec-
tively. Although the potential effects of these mutations
have been previously analyzed by homology modeling
(Banerjee-Basu and Baxevanis, 2004), the accuracy of
these analyses was limited by the divergence of the
FOXP forkhead domain from the rest of the FOX family
and the subtlety of many disease-linked mutations. The
1.9 Å resolution structures of FOXP2 and its complex
with DNA described here allow us to examine these mu-
tations in detail.
The mutation of Arg553 to histidine in human FOXP2
has been linked to a severe congenital speech disorder.
Because of the relatively lower resolution of the previous
FOXA3/DNA crystal structure, the precise role of this
highly conserved arginine residue (Arg168 in FOXA3) in
DNA recognition by the FOX family of transcription fac-
tors has not been clear (Clark et al., 1993). The present
study shows that Arg553 is a major component of the
FOXP2/DNA binding surface (Figure 2A). The guanidi-
nium group of Arg553 forms a water-mediated hydro-
gen bond with Thy110while its long aliphatic side chain
makes extensive van der Waals contacts to Thy110,
Ade120, and Ade130. Thus, the molecular mechanism of
the congenital speech disorder is that the Arg553His
mutation likely disrupts the organization of the protein/
DNA interface in afflicted individuals.
Because of the high sequence identity between
FOXP2 and FOXP3 (Figure 1C), the structure of FOXP2
can also be used to analyze the mutations in FOXP3
linked to human IPEX syndrome. These mutations in-
clude Ile363Val (Ile530 in FOXP2), Phe371Cys and
Phe371Leu (Phe538 in FOXP2), Ala384Thr (Ala551 in
FOXP2), and Arg397Trp (Arg564 in FOXP2). Ile530 is lo-
cated on helix H2 (Figure 6A). Its Cd carbon makes van Figure 6. Disease Mutations
der Waals contact with Leu527, Leu556, and Trp573.
(A) IPEX mutation Ile363Val in FOXP3. The corresponding residue in
Leu527 and Trp573 contact DNA backbone ribose moi- FOXP2, Ile530, forms a cascade of van der Waals interactions with
Leu527, Leu556, and Trp573 that contribute directly or indirectly to
eties directly, while Leu556 holds helix H3 in position to
DNA binding.
recognize DNA. The Ile363Val mutation appears to cause
(B) IPEX mutation Ala384Thr in FOXP3. The corresponding residue
disease by a mechanism wherein it alters DNA binding
Ala551 in FOXP2 on helix H3 packs intimately against Tyr509. The
by influencing neighboring residues that contact DNA.
Ala384Thr mutation would bring an extra g-methyl group into
Thus, a relatively conservative mutation like Ile363Val
a tightly packed protein/DNA interface and disrupt DNA binding.
in FOXP3 can potentially alter its DNA binding and ulti- (C) IPEX mutations Phe371Cys and Phe371Leu in FOXP3. The corre-
sponding residue Phe538 in FOXP2 is critically located at the center
mately lead to autoimmune disease. It is remarkable
of the domain-swapped dimer interface. Mutations of this phenylal-
that a single methyl group, when placed strategically in
anine residue to Cys or even a hydrophobic residue Leu may disrupt
protein, can have such significant impact on biology.
dimerization.
This point is also well-illustrated in the Ala384Thr muta-
tion (Figure 6B). In FOXP2, the corresponding residue
Ala551 on helix H3 is intimately packed against Tyr509, likely destabilize the protein/DNA interaction through
while a serine residue, as seen in many classical FOX several effects including steric repulsion and the loss of
proteins, can be accommodated in this position. Thus, the positive charge.
the Ala384Thr mutation in FOXP3 appears to cause dis- In mutations Phe371Cys and Phe371Leu, the corre-
ease by a mechanism wherein an extra g-methyl group sponding residue Phe538 is exposed in the FOXP2
is forced into a tightly packed protein/DNA interface, monomer structure and plays no apparent role in DNA
thereby disrupting DNA binding. Arg564, located on binding and protein folding (Figure 3D). It is notable
strand S2, binds DNA on the backbone and in the minor that FOXO4 has a valine at this position (Figure 1C), so
groove (Figure 2A). Mutation of this arginine to the bulky a phenylalanine is not required for function here across
and neutral tryptophan residue in FOXP3 (Arg397Trp) will the entire FOX family, arguing that this residue may have
Structure of FOXP2 Bound to DNA
165
(BTP) (pH 6.68), 100 mM NaCl, 6% (w/v) PEG 3000, 10 mM MgCl2,
a specialized function in the FOXP subfamily. Indeed,
and 0.01% (w/v) sodium azide. Typically, crystals grew to approxi-
Phe538 is critically located at the center of the domain-
mately 100 3 100 3 50 mm in 1 2 weeks. Crystals belong to the
swapped dimer interface (Figure 6C). Thus, mutations
space group P21 with cell dimensions a = 67.54 Å, b = 124.21 Å,
Phe371Cys and Phe371Leu in FOXP3 may exert their
c = 67.67 Å, and b = 110.81º.
disease-causing effects by a mechanism wherein the
core of the domain-swapped dimer is disrupted. Con-
Data Collection, Structure Determination, and Analysis
sistent with this observation, mutations disrupting the
Crystals were stabilized in the harvest/cryoprotectant buffer: 50 mM
function of the leucine zipper motif in FOXP3 have also BTP (pH 6.68), 100 mM NaCl, 21% (w/v) PEG 3000, and 25% (w/v)
glycerol and flash frozen with liquid nitrogen for cryocrystallogra-
been linked to IPEX (Chatila et al., 2000). Overall, the
phy. Data were collected at the ALS BL8.2.1 beamline at the Law-
high-resolution crystal structure of the FOXP2/DNA
rence Berkeley National Laboratory and BioCARS sector 14-BM-C
complex provides a structural basis for understanding
of Advanced Photon Source, Argonne National Laboratory. Data
disease-causing mutations in FOXP2 and FOXP3, which
were reduced using DENZO and SCALEPACK (Otwinowski and
in turn support the physiological relevance of our struc-
Minor, 1997). Initial phases were determined by molecular replace-
tural and biochemical observations. ment using the coordinates of FOXA3 (Clark et al., 1993). Molecular
replacement, refinement, and final analysis were done with CNS
In summary, high-resolution structural studies of the
(Brunger et al., 1998). Final models have very good geometry. All res-
FOXP2/DNA complex reported here have revealed the
idues are in the allowed regions of the Ramachandran plot, with 92%
general mechanism of DNA recognition by the FOX
in the most favored regions. The statistics of crystallographic anal-
family of transcription factors and unusual biochemical
ysis are presented in Table 1. Figures of structure illustration were
properties of the FOXP2 forkhead domain. The most
prepared using MOLSCRIPT (Kraulis, 1991) and Pymol (DeLano Sci-
surprising finding from these studies is that the FOXP2 entific, San Francisco, CA). Model building and structural compari-
sons were carried out in O (Jones et al., 1991).
forkhead domain can form a domain-swapped dimer.
Disease-related mutations, sequence comparison, and
Mutagenesis and Multiangle Light Scattering Analysis
biochemical analyses argue strongly that this domain
The Ala539Pro mutation in human FOXP2 (amino acids 503 584) was
swapping is a physiologically relevant function evolved
made by site-specific mutagenesis (QuikChange; Stratagene, La
in the P branch of FOX proteins. Though the biological
Jolla, CA). The mutant was expressed and purified similarly to the
significance of swapping in FOXP2 has yet to be deter-
wild-type protein (see above). Both the mutant and the wild-type pro-
mined, swapping is thought to have functional roles in
tein were analyzed by a multiangle light scattering detector (DAWN-
several other proteins. The most notable examples are EOS; Wyatt Technologies, Santa Barbara, CA) after separation by
found in the cadherins, which utilize an exchange of N- a size exclusion column (KW-803; Shodex, Aston, PA) on HPLC.
terminal b strands to mediate dimerization in cell-cell
Acknowledgments
adhesion (Chen et al., 2005; Shapiro et al., 1995). Unlike
most dimeric transcription factors that bind adjacent
The authors thank Liang Guo, Luke G. Kroiss, Andrew Bonham, Greg
DNA sites, modeling predicts that domain-swapped
Smith, and Keith Brister for help in data collection and discussions;
FOXP dimers can only bind cognate DNA sites sepa-
Youngchang Kim, James Whisstock, Ning Lei, Pearl Quartey, and
rated far from each other or located on different DNA
Frank Collart for assistance in initial protein expression, purification,
strands, suggesting that a specialized function of FOXP and bioinformatics analysis; and Jim Goodrich, Marcelo Sousa, Deb-
bie Wuttke, and Mitchell Guss for critical reading of the manuscript.
proteins in transcriptional regulation is to promote the
H.T. was supported by the Australian Synchrotron Research Pro-
assembly of higher order protein/DNA complexes. These
gram (ASRP). The use of the BioCARS sector at the Advanced Photon
insights will facilitate further studies of the role of FOXP2
Source was supported by ASRP, NIH/National Center for Research
in human language development and FOXP3 in immuno-
Resources, and the U.S. Department of Energy. This research is sup-
logical tolerance.
ported by grants from the W.M. Keck Foundation (L.C.) and NIH (L.C.).
Received: August 3, 2005
Experimental Procedures
Revised: September 27, 2005
Accepted: October 4, 2005
Sample Preparation and Crystallization
Published: January 10, 2006
The forkhead domain of human FOXP2 (amino acids 503 584) was
cloned into the pET-30 LIC vector as a 63 histidine-tagged fusion
protein and was expressed in Rosetta pLysS cells (Novagen, San
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