From the Skirball Institute of Biomolecular Medicine
and Department of Pharmacology, New York University School of
Medicine, New York, New York 10016 and the § Laboratory of
Molecular Biology, NIDDK, National Institutes of Health, Bethesda,
Maryland 20892
Received for publication, November 25, 2002, and in revised form, January 19, 2003
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ABSTRACT |
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Grb7, Grb10, and Grb14 are members of a
distinct family of adapter proteins that interact with various receptor
tyrosine kinases upon receptor activation. Proteins in this family
contain several modular signaling domains including a pleckstrin
homology (PH) domain, a BPS (between PH
and SH2) domain, and a C-terminal Src homology 2 (SH2)
domain. Although SH2 domains are typically monomeric, we show that the
Grb10 SH2 domain and also full-length Grb10 Grb10 is a member of a family of adapter proteins including Grb7
and Grb14, which have been identified as putative downstream effectors
of receptor tyrosine kinases (1). Proteins in this family contain
several modular domains including an N-terminal proline-rich region, a
Ras-associated-like domain, a pleckstrin homology
(PH)1 domain, a short region
known as the BPS (between PH and
SH2) or PIR (phosphorylated insulin
receptor-interacting region) domain, and a
C-terminal Src homology 2 (SH2) domain. Grb10 has multiple alternatively spliced forms ( The BPS/PIR and SH2 domains of Grb7/Grb10/Grb14 have been
implicated in the interaction with activated receptor tyrosine kinases. The BPS domain is a structurally uncharacterized region of ~50 residues that is unique to this family of adapter proteins. The SH2
domain, found in many signaling proteins, is a well characterized protein module of ~100 residues that binds phosphotyrosine-containing sequences (2, 3). The BPS domains of Grb7/Grb10/Grb14 bind to the
phosphorylated insulin and insulin-like growth factor-1 (IGF1)
receptors (4-6). The SH2 domains of Grb7/Grb10/Grb14 also interact
with the insulin and IGF1 receptors (4, 5, 7-11) and, in addition,
have been shown to mediate the interaction with the epidermal growth
factor receptor (4, 12), Her2 (epidermal growth factor receptor family)
(13, 14), platelet-derived growth factor receptor- The roles of Grb10 family members in growth factor-mediated
signaling have not been clearly established. Most studies have investigated the effects of these adapter proteins on mitogenesis. Several studies have indicated that Grb10 and Grb14 negatively regulate
insulin- and IGF1-mediated mitogenic signaling (5, 7, 8, 22), possibly
through direct inhibition of receptor catalytic activity by the BPS
domain (23, 24). In contrast, Grb10 has been reported to enhance
platelet-derived growth factor-stimulated mitogenesis (16). Moreover,
in many human breast cancer cell lines, Grb7 is co-overexpressed with
and bound to Her2 and thus might potentiate Her2 signaling (13).
To begin to understand the structural basis for the
interaction of Grb10 with the insulin and IGF1 receptors, we have
determined the crystal structure of the SH2 domain of Grb10. Although
SH2 domains typically function as monomers, gel filtration and
sedimentation equilibrium studies show that the Grb10 SH2 domain and
full-length Grb10 Production of Grb10 Proteins--
Both wild-type and mutant
(Phe-515
The isolated Grb10 SH2 domains, wild type (SH2WT), and
Phe-515 Crystallization and X-ray Analysis--
Crystals of the Grb10
SH2 domain were grown by vapor diffusion at 4 °C using the hanging
drop method. 2 µl of protein solution (4 mg/ml in 20 mM
HEPES, pH 7.5, and 100 mM NaCl) was mixed with 2 µl of a
crystallization buffer (10% polyethylene glycol 8000, 100 mM HEPES, pH 7.5, and 5 mM
Tris[2-carboxyethyl]phosphine, and the drop was suspended over
500 µl of the crystallization buffer and allowed to equilibrate for 2 days. The equilibrated drops were then suspended over a reservoir
buffer that contained 15% polyethylene glycol 8000. The crystals
belong to the monoclinic space group P21 with unit cell
dimensions a = 28.82 Å, b = 49.04 Å,
c = 79.61 Å, and Gel Filtration Chromatography and Gel-shift Assays--
Gel
filtration chromatography was performed on a Superose-12 10/30 column,
which was equilibrated in 20 mM HEPES, pH 7.5, 100 mM NaCl, and 1 mM dithiothreitol for all Grb10
samples and protein standards. The column flow rate was 0.4 ml/min. For
the gel-shift studies, 1 µl of purified BPS-SH2WT or
BPS-SH2F515R at 200 µM was mixed with 1 µl
of the tris-phosphorylated insulin receptor kinase domain (IRK)
(residues 978-1283) at 100 µM and prepared essentially
as described by Hubbard (32). Mixtures were loaded onto a 20% native
PhastGel and electrophoresed with the PhastGel system (Amersham
Biosciences). The gels were stained with PhastGel Blue R.
Sedimentation Equilibrium Ultracentrifugation--
Samples of
the Grb10 SH2 domain were prepared in 50 mM HEPES, pH 7.5, and 100 mM NaCl and analyzed at loading concentrations of
A280 = 0.10, 0.21, 0.28, 0.41, and 0.51. Sedimentation equilibrium experiments were conducted at 4.0 °C in a
Beckman Optima XL-A analytical Ultracentrifuge. Samples (loading volume
of 160 µl) were studied at different rotor speeds corresponding to
12,000, 14,000, 16,000, and 18,000 rpm. Data were acquired as an
average of 8 absorbance measurements at a nominal wavelength of 280 nm and a radial spacing of 0.001 cm. Equilibrium was achieved within 48 h. Data were initially analyzed in terms of a single ideal solute to obtain the buoyant molecular mass,
M1(1 Overall Description of the Grb10 SH2 Domain Structure--
The
crystal structure of the SH2 domain of Grb10
The dimer interface comprises residues from the C-terminal half of the
SH2 domain (Fig. 2B), primarily in Absence of a P+3 Binding Pocket and Aberrant BC Loop in the Grb10
SH2 Domain--
Previous structural studies have shown that
phosphotyrosine-containing peptides generally bind in an extended
conformation perpendicular to the
A superposition of the Grb10 SH2 domain and the Src SH2 domain with
bound phosphopeptide (36) indicates that the binding pocket for the P+3
residue in the Grb10 SH2 domain is absent (Fig. 4A) because of Val-522 in the
BG loop (BG3) (Fig. 4B). This residue along with Asp-500 in
the EF loop (EF1) seals off the peptide binding cleft at the P+3
position. Asp-500 is hydrogen-bonded to both the backbone and side
chain of Thr-504 (Fig. 2B), a residue in the dimer
interface, as well as to the side chain of Ser-498. Val-522 is
conserved in Grb14 and is an isoleucine in Grb7. The BG loop of Grb10
is relatively short and of the same length as the BG loops in Src, Abl,
and Zap70. In these other SH2 domains, a glycine is present at the
position of Val-522 (BG3); lack of a side chain at this position
provides the pocket for the P+3 residue of the phosphopeptide.
The BC loop in SH2 domains interacts with the phosphate group of the
phosphopeptide. A majority of SH2 domains possess a five-residue BC
loop, and nearly all of these contain a glycine at the end of the loop
(BC5) including Src, Shp2, Abl, and Grb2. The BC loop in the SH2
domains of Grb7/Grb10/Grb14 is also five residues long, but it ends
with lysine (Grb10/14) or glutamine (Grb7) rather than glycine. The
backbone dihedral angles ( Solution Studies of the Grb10 SH2 Domain and Full-length
Grb10
As shown in Fig. 5A, the
wild-type Grb10 SH2 domain (molecular mass = 12.4 kDa) elutes at
a volume consistent with a dimer, whereas the Phe-515
An analysis by gel filtration chromatography of full-length Grb10 Interaction of Grb10 BPS-SH2 with the Phosphorylated Insulin
Receptor Kinase--
Interaction of full-length Grb10 with the insulin
receptor involves both the SH2 domain and the BPS domain (4). We have previously shown that the purified Grb10 BPS-SH2 protein binds to the
tris-phosphorylated form of the insulin receptor kinase domain
(IRK3P), but not to the unphosphorylated form (23). We
analyzed binding of wild-type Grb10 BPS-SH2 (dimeric) or mutant BPS-SH2
(Phe-515 The crystal structure of the Grb10 SH2 domain reveals a 2-fold
symmetric dimer with residues in and flanking the C-terminal SH2 domains are typically monomeric, both in solution and in crystal
structures. Two crystal structures of dimeric SH2 domains have been
reported previously. In a crystal structure of the SH2 domain of Shc
(37), a disulfide-bonded dimer was observed, which would not be favored
in the reducing environment of the cytosol. Moreover, the Shc SH2
domain is monomeric in solution at physiologic pH (37). A
domain-swapped dimeric form of the Grb2 (unrelated to Grb7/Grb10/Grb14)
SH2 domain has been observed crystallographically (38), as has a
monomeric form (39), but this dimer is likely to be an artifact of
expression as a glutathione S-transferase fusion protein
(40).
From gel filtration and sedimentation equilibrium experiments (Fig. 5),
the Grb10 SH2 domain is dimeric in solution at physiologic pH with a
Kd of ~2 µM. Substitution of Phe-515
in the dimer interface with arginine (or alanine, data not shown)
yields a monomeric SH2 domain. This provides strong evidence that the crystallographic dimer is also the solution dimer. Whether full-length Grb10 A previous study (42) on the oligomerization state of Grb10 has been
reported in which it was concluded that Grb10 The crystal structure of the Grb10 SH2 domain indicates that the
binding pocket for the P+3 residue of a phosphopeptide
ligand is absent because of a valine (Val-522) in the BG loop (BG3)
rather than a glycine (Fig. 4, A and B), which is
found at this position in the SH2 domains of, for example, Src and Abl
(Fig. 3). In contrast to the N-terminal SH2 domain of p85 in which
rotation of the Tyr-416 (BG5) side chain accommodates binding of the
P+3 residue (43), a simple rotation of the (branched) Val-522 side
chain in the Grb10 SH2 domain will not unmask a P+3 binding pocket. In
addition to Val-522, the side chain of Asp-500 in the EF loop (EF1)
extends into the space between the EF and BG loops in which the P+3
residue would normally bind (Fig. 4B). The side chain
rotamer of Asp-500, a conserved residue in the Grb7/Grb10/Grb14 family,
is stabilized through hydrogen bonding to several residues including
Thr-504 ( Dimerization of the Grb10 SH2 domain is likely to fortify the positions
of the EF and BG loops and thus contribute to ligand specificity.
Although the residues in the interface of the Grb10 SH2 dimer are
conserved in Grb7/Grb14, other residue differences in the SH2 domains
of these proteins could affect the strength of the dimer interface. For
example, a tyrosine in Grb10/Grb14 at position Precedence for the lack of a P+3 binding pocket comes from the SH2
domain of Grb2, which shows strong specificity for phosphopeptides containing an asparagine at the P+2 position but no preference at the
P+3 position (44). The crystal structure of the Grb2 SH2 domain shows
that the bulky Trp-121 in the EF loop (EF1) forces the phosphopeptide
to turn away from the SH2 domain at the P+2 position, facilitated by
hydrogen bonds involving Asn(P+2) (39). Numerous studies have indicated
that the SH2 domain of Grb7, like that of Grb2, has a preference for
phosphopeptides with the turn-facilitating asparagine at the P+2
position (14, 15, 19, 20, 45), e.g. pTyr-1139
(pYVNQ) in Her2 (14). Thus, the hydrophobic residue in the
BG loop, valine (Val-522) in Grb10/14 or isoleucine in Grb7, appears to
function similarly to Trp-121 in the EF loop of Grb2,
i.e. to discriminate against extended phosphotyrosine sequences and select for sequences that are predisposed to turn away
from the surface of the SH2 domain after the phosphotyrosine.
In addition to influencing ligand specificity (through EF and BG loop
stabilization), dimerization of the Grb10 SH2 domain would favor the
binding of dimeric ligands such as the two Differences in the phosphopeptide-binding properties of the
Grb7/Grb10/Grb14 SH2 domains have been reported previously (5, 6, 24).
With respect to binding to the phosphorylated insulin receptor, the SH2
domain of Grb7 appears to bind with the highest affinity followed by
Grb10 and then Grb14 (6). In addition, the Grb7 SH2 domain interacts
relatively strongly with pTyr-1139 (pYVNQ) in Her2 compared with the
Grb14 SH2 domain (14). In the study by Janes et al. (14),
selected Grb14 residues were swapped for the corresponding Grb7
residues and binding of the mutant Grb14 SH2 domains to pTyr-1139 in
Her2 was measured. One of the key residues pinpointed was A non-glycl residue at the end of the five-residue BC loop of
Grb7/Grb10/Grb14 family members, rare in SH2 domains, alters the
conformation of the BC loop (Fig. 4C). Consequently, the
residue difference between Grb7 and Grb10/Grb14 at BC2 (Ser-466 in
Grb10) is probably also important in the tighter association of Grb7 with phosphopeptides. An arginine at this position in Grb7 is likely to
interact with the phosphate group of the phosphotyrosine, whereas the
shorter serine in Grb10/Grb14 might not directly coordinate the
phosphate group. In SH2 domains with a glycine at BC5, the corresponding serine does hydrogen bond with the phosphate group (Fig.
4C). In addition, one or two hydrogen bonds typically made between the phosphate group and backbone nitrogen atoms in the tip of
the BC loop could be compromised because of the non-glycl residue at
BC5 in Grb7/Grb10/Grb14.
Based on the structural results presented here and biochemical results
reported previously (4-6), we propose that the SH2 domains of
Grb10/Grb14 are partially impaired in their ability to bind
phosphotyrosine-containing ligands because of the non-glycl residue at
the end of the BC loop and the lack of a P+3 binding pocket. Amino acid
substitutions in the Grb7 SH2 domain such as leucine at One plausible explanation for the reduced ligand binding capabilities
of the Grb10/Grb14 SH2 domains is that the BPS domain, which is unique
to this adapter family, also contributes to the interaction with the
insulin and IGF1 receptors (4-6). Too high an overall affinity would
alter the balance between Grb10/Grb14 binding and the binding of other
downstream proteins. Indeed, isothermal titration calorimetry data (not
shown) indicate that the tandem BPS-SH2 domains of Grb10 tightly
associate with the phosphorylated insulin receptor kinase domain (IRK)
(Kd <100 nM).
The relative contributions of the BPS and SH2 domains of
Grb7/Grb10/Grb14 to insulin receptor binding varies. For Grb7, the interaction is mediated primarily by the SH2 domain (6), whereas for
Grb14, the BPS domain predominates (5). In contrast, the two domains in
Grb10 contribute approximately equally (4). Thus, engagement of Grb10
with the activated insulin receptor would appear to involve two modest
affinity interactions that, in combination, provide high affinity and specificity.
The binding sites on the insulin receptor for the SH2 and BPS domains
of Grb10 have not been unambiguously identified. In the context of
full-length Grb10 (i.e. with the BPS domain present), the
SH2 domain interacts with the phosphorylated activation loop of the
insulin receptor (4, 11). The BPS domain also interacts with the core
kinase domain, and, like the SH2 domain, binding requires
phosphorylation of the activation loop (4). Yet from peptide
competition experiments, the BPS domain does not appear to bind
directly to the activation loop (23). The BPS domains of Grb10/14 have
been shown to inhibit the catalytic activity of the insulin and IGF1
receptors (23, 24) in a manner that is non-competitive with ATP and
non-competitive with substrate peptide (24). These data suggest that
the BPS domain binds to a site on IRK distal to the active site, which
becomes accessible only upon activation loop phosphorylation.
Based on the aforementioned studies and our gel-shift results that
indicate formation of a 2:2 complex between Grb10 BPS-SH2 and
phosphorylated IRK (Fig. 6A), we propose the following
interaction model for Grb10 are dimeric in solution
under physiologic conditions. The crystal structure of the Grb10 SH2
domain at 1.65-Å resolution reveals a non-covalent dimer whose
interface comprises residues within and flanking the C-terminal
helix, which are conserved in the Grb7/Grb10/Grb14 family but
not in other SH2 domains. Val-522 in the BG loop (BG3) and Asp-500 in
the EF loop (EF1) are positioned to interfere with the binding of the
P+3 residue of a phosphopeptide ligand. These structural
features of the Grb10 SH2 domain will favor binding of dimeric,
turn-containing phosphotyrosine sequences, such as the phosphorylated
activation loops in the two
subunits of the insulin and
insulin-like growth factor-1 receptors. Moreover, the structure
suggests the mechanism by which the Grb7 SH2 domain binds selectively
to pTyr-1139 (pYVNQ) in Her2, which along with Grb7 is co-amplified in
human breast cancers.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
), which differ in their
N-terminal region including the PH domain.
(10, 15, 16), Ret
(17), EphB1 (18), Kit (19), Tie2 (20), and fibroblast growth factor receptor-1 (21).
are dimeric in solution. The crystal structure
reveals the molecular basis for dimerization of the Grb10 SH2 domain. The dimer interface comprises residues in the C-terminal half of the
domain, which are conserved in the Grb7/Grb10/Grb14 family. Several
sequence and structural features of the Grb10 SH2 domain including its
mode of dimerization are predicted to discriminate against binding of
canonical, extended phosphotyrosine-containing sequences and favor
binding of turn-containing sequences.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Arg) full-length human Grb10
(residues 1-536) and
BPS-SH2 constructs (residues 357-536) were subcloned into expression
vector pET21 (Novagen). All of the constructs were verified by DNA
sequencing. Escherichia coli strain BL21(DE3) was
transformed with the plasmids, and cultures were grown in Luria Broth
at 37 °C to an A600 of 0.8. Protein expression was induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside (1.0 mM final) for 3 h at 30 °C. Bacteria were harvested
by centrifugation, resuspended in lysis buffer (20 mM
Hepes, pH 7.5, 0.1% Triton X-100, 1 mM PMSF, and Complete
protease inhibitor tablets (Roche Molecular Biochemicals)), and lysed
by French press. The lysate was centrifuged at 20,000 × g for 1 h, and the supernatant was collected. Proteins
were purified from the soluble fraction using cation-exchange
chromatography (Fractogel, EM Science) followed by gel filtration
chromatography (Superdex-75, Amersham Biosciences).
Arg mutant (SH2F515R) were obtained by mixing
purified BPS-SH2 (4 mg/ml final) at room temperature with elastase (10 µg/ml final) for 2.5 h. The resulting proteolyzed fragments were
purified by gel filtration chromatography (Superose-12, Amersham
Biosciences). A 12,340-Da fragment was verified to be the SH2 domain
(residues 429-533) by mass spectrometry and N-terminal sequencing.
Protein concentrations were determined using calculated extinction
coefficients (25).
= 96.62°. There are two
molecules of the SH2 domain in the asymmetric unit yielding a solvent
content of ~46%. Crystals were equilibrated in a cryoprotectant of
reservoir buffer plus 10% glycerol before being flash-frozen in liquid
propane. Data were collected at beamline X12C at the National
Synchrotron Light Source, Brookhaven National Laboratory. The data were
processed using Denzo and Scalepack (26). A molecular replacement
solution was found with AMoRE (27) using a homology model (28) of the Grb10 SH2 domain based on the crystal structure of the N-terminal SH2
domain of Syk (Protein Data Bank code 1A81) (29). Simulated annealing,
rigid-body, positional, and B-factor refinement were carried out using
CNS (30). Model building was performed with O (31). Buried surface area
in the dimer interface was calculated with CNS (30) using a probe
radius of 1.4 Å.
v1
), using
the Optima XL-A data analysis software (Beckman). The values of
M1 were calculated using densities (
) at
4.0 °C obtained from standard tables and a calculated
v1 = 0.7389 ml/g (33). Data from sedimentation
equilibrium experiments performed at loading concentrations of
A280 = 0.21 and 0.10 were analyzed in terms of
reversible monomer-dimer equilibria, essentially as described by
Jenkins et al. (34).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was solved by molecular
replacement using a homology model (28) based on the N-terminal SH2
domain of Syk (29) and was refined at 1.65-Å resolution. A
2Fo
Fc electron density map is
shown in Fig. 1, and the crystallographic
statistics are given in Table I. The
overall structure of the Grb10 SH2 domain is similar to those of
other SH2 domains (2), possessing a core anti-parallel
sheet
flanked on either side by an
helix (Fig.
2A). The asymmetric unit
comprises two SH2 domains that form a non-covalent dimer with the two
protomers related by a molecular 2-fold axis. The atomic model includes
residues 429-533 with the exception of residues 490-494 (DE loop),
which are disordered in both molecules. The overall root mean square
deviation in the C
positions in the two protomers is 0.5 Å.
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Fig. 1.
Electron density map in the Grb10 SH2
dimer interface. The 2Fo Fc electron density map (1.65-Å resolution, 1
contour) is shown as wire mesh (cyan), and the two SH2
domain protomers (after refinement) are shown in a stick
representation colored in gold and
purple. Ordered water molecules are indicated with red
crosses. Selected residues are labeled. An apostrophe
in the label differentiates residues in the purple protomer
from the gold protomer.
X-ray data collection and refinement
statistics
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Fig. 2.
Crystal structure of the Grb10 SH2
dimer. A, ribbon diagram of the Grb10 SH2
dimer. The two individual SH2 domain protomers are colored
gold and purple. Secondary structure elements are
labeled using the nomenclature described previously (35), and labeling
for the two protomers is differentiated by the use of an
apostrophe for the second (purple) protomer. The
N and C termini are indicated by N and C. The
BC, EF, and BG loops are explicitly
labeled, and the disordered DE loops (residues 490-494) are
depicted with broken lines. The non-crystallographic 2-fold
axis is perpendicular to the page. B, stereoview of the
dimer interface. The orientation and coloring of the protomers is the
same as in A. The backbone of the protein is shown in a
coil representation. Backbone and side chain atoms in the
dimer interface are shown in ball-and-stick representation.
Hydrogen bonds are represented by dashed black lines, and
residues making hydrophobic contacts are shown with semi-transparent
van der Waals surfaces. Carbon atoms are colored either
gold or purple, oxygen atoms are shown in
red, and nitrogen atoms are in blue.
-helix B (
B), and
is a composite of hydrophobic and hydrophilic interactions. In the
middle of the interface is Phe-515 (
B8; SH2 domain nomenclature is
from Eck et al. (35)), which is packed against Phe-515 and Thr-504 (
F1) from the other protomer (designated Phe-515' and Thr-504'). The side chain of Gln-511 (
B4) is hydrogen-bonded to the
backbone of Asp-514' as well as to the backbone of Asp-508 and the side
chain of Ser-507. The side chain of Asn-519 (
B12) makes two hydrogen
bonds to the backbone of Lys-505'. The interface is capped on each end
through packing of Leu-518 (
B11) with Phe-496' (
E1). The total
surface area buried in the interface is 780 Å2 (~390
Å2/protomer, representing 6.6% of the surface area). A
sequence alignment of the SH2 domains of Grb7/Grb10/Grb14 (Fig.
3) shows that the residues in the Grb10
SH2 dimer interface are strictly conserved with the exception of
Phe-496, which in Grb7 is conservatively substituted with tyrosine.
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Fig. 3.
Structure-based sequence alignment of the SH2
domains of Grb7, Grb10, and Grb14. The sequences are for the human
proteins. A dot (·) in the alignment indicates sequence
identity to Grb10. The location and length of the secondary structure
elements as determined from the Grb10 crystal structure are shown above
the alignment with helices indicated by "h,"
strands indicated by "s," and disordered residues
indicated by "~." Residues in the dimer interface are shown in
green. Residues in Grb10 that are poised to block binding of
the P+3 residue of a phosphopeptide are shown in red.
Residues that are predicted to affect phosphate coordination by the BC
loop are shown in blue. The canonical SH2 domain secondary
structure assignments (35) are shown below the alignment. Orange
boxes represent
strands, and yellow boxes represent
helices. For example, BC2 refers to the second residue in the BC
loop (counting in the bottom boxes), which is Ser-466 in
Grb10, and
B8 refers to the eighth residue in
B, which is Phe-515
in Grb10.
sheet of the SH2 domain (2). In a
typical SH2-phosphopeptide interaction, two deep pockets in the SH2
domain engage the phosphotyrosine (P residue) and the P+3 residue of the phosphopeptide (36), which is often hydrophobic. The binding pocket
for phosphotyrosine comprises residues from
A,
B,
D, and the
BC loop. An invariant arginine (
B5; Arg-462 in Grb10) salt-bridges
with the phosphate moiety of the phosphotyrosine. The binding pocket
for the P+3 residue is formed between the EF and BG loops.
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Fig. 4.
Absence of a P+3 binding pocket and aberrant
BC loop in the Grb10 SH2 domain. A, surface
representations of the Src SH2 domain with bound pYEEI phosphopeptide
(left) (36) and of one of the Grb10 SH2 domain protomers
(right). The phosphopeptide is shown in a red stick
representation. The orientations of the two SH2 domains are the
same, obtained through C superposition of the core
sheet. The
surface coloring is according to curvature: green, high
convex curvature; black, high concave curvature. The binding
pocket for the P+3 residue of the phosphopeptide (isoleucine) in the
Src SH2 domain is indicated by a dashed yellow circle. The
corresponding location in the Grb10 SH2 domain is also indicated by a
dashed yellow circle, which shows that the P+3 binding
pocket is absent in the Grb10 SH2 domain. B, backbone
representation of the Grb10 SH2 domain (same orientation/scale as in
right panel of A) with residues Asp-500 (EF1) and
Val-522 (BG3) responsible for the lack of a P+3 binding pocket shown in
a red stick representation. C, stereoview of a
superposition between the BC loops of the Grb10 SH2 domain and the LCK
SH2 domain (35). Bonds and carbon atoms of Grb10 and LCK are
colored gold and gray, respectively. Oxygen atoms
are colored red, nitrogen atoms are blue, and
phosphorus atoms are black. Only selected (labeled) side
chains are shown. Others are truncated to C
. The residues in LCK corresponding to the labeled residues in Grb10 are Arg-154
(Arg-462 in Grb10), Ser-156 (Ser-464), Ser-158 (Ser-466), Ala-160
(Pro-468), and Gly-161 (Lys-469). The black arrow points to
the major difference in backbone conformation in the BC loop between
Pro-468/Lys-469 in Grb10 and Ala-160/Gly-161 in LCK. Hydrogen bonds to
the phosphotyrosine moiety in the LCK structure are shown as
black dashed lines.
,
) for this glycine in the structure
of the ligand-bound LCK (Src family) SH2 domain (35) are (108 and
17°), a combination that is much less energetically favorable
for a residue with a side chain. In the Grb10 SH2 domain structure, the
backbone dihedral angles of Lys-469, particularly
, are
substantially different (
114 and 6°), resulting in a difference in
conformation of the BC loop (Fig. 4C). The non-glycl residue at BC5 in Grb7/Grb10/Grb14 is likely to impair phosphate coordination by the BC loop.
--
During purification, the Grb10 SH2 domain eluted from a
gel filtration column at a volume corresponding to a dimer,
consistent with the crystallographic results. Based on the key position
of Phe-515 at the center of the dimer interface (Figs. 1 and
2B), mutant Grb10
proteins (SH2 domain and full-length)
were generated with the substitution Phe-515
Arg, which would be
predicted to yield a monomeric SH2 domain. The oligomeric properties of the purified wild-type and mutant proteins were examined by gel filtration chromatography and sedimentation equilibrium ultracentrifugation.
Arg mutant
elutes at a volume corresponding to a monomer. Sedimentation
equilibrium experiments were performed to further analyze the
oligomeric properties of the wild-type SH2 domain. At a high loading
concentration (A280 = 0.28, ~40 µM), an analysis in terms of a single ideal solute
yielded buoyant molecular masses that were independent of the rotor
speed, indicating that the sample was monodisperse. The experimentally
determined molecular mass of 26,310 ± 480 g/mol shows that the
SH2 domain is dimeric in solution. At lower loading concentrations
(A280 = 0.10 and 0.21), modeling of the data in
terms of a single ideal solute returned buoyant masses lower than that
expected for a dimer but larger than that expected for a monomer. Data
for the two concentrations were analyzed globally in terms of a
reversible monomer-dimer equilibrium. Excellent fits were obtained
(Fig. 5B) yielding Kd values of 1.0 ± 0.4 µM (A280 0.21) and 2.8 ± 0.5 µM (A280 0.10). Therefore,
the Grb10 SH2 domain undergoes reversible dimerization with a
Kd of ~2 µM.
View larger version (21K):
[in a new window]
Fig. 5.
Solution studies of the Grb10 SH2 domain and
full-length Grb10 . A, left,
100 µl of the wild-type (black) or mutant
(Phe-515
Arg) (gray) Grb10 SH2 domain were loaded onto
a Superose-12 size-exclusion column at a concentration of ~250
µM for each; right, 100 µl of wild-type
(black) or mutant (Phe-515
Arg) (gray)
full-length Grb10
were loaded onto a Superose-12 column at 40 or 50 µM, respectively. The elution positions of standards are
indicated by dashed vertical lines (catalase (232 kDa);
aldolase (158 kDa); albumin (67 kDa); ovalbumin (43 kDa);
chymotrypsinogen A (25 kDa); and ribonuclease A (14 kDa)).
B, sedimentation equilibrium profiles at 280 nm
(bottom) and the corresponding residuals (top)
for the Grb10 SH2 domain at 16,000 (triangles) and 12,000 (circles) rpm and 4.0 °C. The initial loading
concentration corresponds to an A280 of 0.21. The lines (gray) through the data represent the
best fit for a reversible monomer-dimer equilibrium. The fitting yields
a ln(Ka) value of 13.8 ± 0.3, corresponding to
a Kd of 1.0 ± 0.4 µM. The
fitting of data (data not shown) for samples at an initial loading
concentration of A280 = 0.10 yielded a
Kd of 2.8 ± 0.5 µM.
(molecular mass = 60.8 kDa) indicates that Grb10
is also oligomeric in solution with an elution volume just greater (lower molecular mass) than that of the 232-kDa molecular mass standard (Fig.
5A). This elution position suggests either a compact
tetramer or an elongated dimer. A trimer is unlikely because of the
observed 2-fold symmetry of the SH2 domain. The full-length Grb10
mutant (Phe-515
Arg) elutes at the same volume as the 67-kDa
standard, consistent with a monomeric protein.
Arg; monomeric) to IRK3P by native-gel
electrophoresis (Fig. 6A). The
gel-shift assay shows that both wild-type and mutant BPS-SH2 form a
complex with IRK3P, but the mobility (a function of size,
charge, and shape) of the mutant complex is significantly greater than
the mobility of the wild-type complex. A change in oligomeric state
notwithstanding, the addition of one positive charge
(Phe-515
Arg) to the protein complex would reduce mobility in the
native gel. These gel-shift results are consistent with formation of a
2:2 complex between wild-type BPS-SH2 and IRK3P and a 1:1
complex between mutant BPS-SH2 and IRK3P.
View larger version (37K):
[in a new window]
Fig. 6.
Interaction between Grb10 BPS-SH2 and
tris-phosphorylated IRK. A, native gel analysis
of complex formation between tris-phosphorylated IRK
(IRK3P) and either wild-type dimeric BPS-SH2
(BPS-SH2WT) or mutant (Phe-515 Arg) monomeric
BPS-SH2 (BPS-SH2F515R). The individual BPS-SH2
proteins (wild type and mutant, lanes 1 and 2) do
not migrate into the gel because of their high pI, whereas
IRK3P alone migrates with high mobility (lane
3). The protein complex produced by mixing BPS-SH2WT
with IRK3P (lane 4) has a slower mobility than
the complex between BPS-SH2F515R and IRK3P
(lane 5), suggesting a 2:2 stoichiometry for
BPS-SH2WT + IRK3P and a 1:1 stoichiometry for
BPS-SH2F515R + IRK3P. B, model for the
interaction of Grb10
with the insulin receptor. The Grb10
SH2
dimer binds to the activation loops of the two tyrosine kinase domains
of the insulin receptor
subunits, facilitated by binding of the PH
domain to phosphatidylinositol phosphates in the plasma membrane
(spheres). The BPS domain of Grb10
binds to an unidentified site on
the kinase domain. The extracellular portion of the insulin receptor is
not shown, nor is the Grb10
Ras-associated-like domain, which is
N-terminal to the PH domain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helix
(
B) mediating the interaction between the two protomers (Fig. 2).
All of the residues in the dimer interface are conserved in the
Grb7/Grb10/Grb14 family with the exception of the conservative substitution of Phe-496
Tyr in Grb7 (Fig. 3). Inspection of a
sequence alignment of SH2 domains indicates that only members of this
adapter family contain hydrophobic residues at positions
E1
(Phe-496),
B8 (Phe-515), and
B11 (Leu-518), suggesting that the
observed mode of dimerization of the Grb10 SH2 domain is unique to this family.
is an elongated dimer or a compact tetramer is not established unequivocally from the gel filtration experiments. The observation that
the point mutation Phe-515
Arg yields a monomeric protein rather
than an intermediate dimeric protein suggests that Grb10
is an
elongated dimer. This supposition is plausible given the multidomain
composition of Grb7/Grb10/Grb14 (Ras-associated-like, PH, BPS, and
SH2). The gel filtration data indicate that oligomerization is mediated
wholly or in large part by SH2 dimerization, although our data do not
rule out the possibility that other regions in Grb10
make minor
contributions to oligomerization. A modest Kd of
~2 µM for SH2 domain dimerization implies that,
depending on cellular levels of Grb10
, the protein could be
monomeric in the cytosol. The PH domain of Grb10
is likely to
interact with phosphatidylinositol phosphates as has been demonstrated
for the Grb7 PH domain (41). Localization of Grb10
to the plasma
membrane via the PH domain will increase the effective protein
concentration, promoting SH2 domain-mediated dimerization of
Grb10
.
/
is a tetramer. In
that study, a CHO cell lysate containing transfected Grb10
(called
Grb10
in Ref. 42) or Grb10
(called Grb10
) was run on a gel
filtration column and Grb10
/
was detected by Western blot
analysis. Our gel filtration and sedimentation equilibrium experiments
were performed with highly purified Grb10
at relatively high
concentrations, which would favor higher order oligomerization, from
which we conclude that Grb10
is most likely to be a dimer. Also in
the study by Dong et al. (42), yeast two-hybrid experiments were performed to identify which domains of Grb10
are responsible for oligomerization. These data indicated that the N-terminal region
interacts with both the PH domain and the BPS-SH2 tandem domains and
that BPS-SH2 does not self-associate. The latter result would seem to
conflict with our crystallographic and solution findings. One possible
explanation for some of these discrepancies is that Grb10
has a
58-residue extension on its N terminus versus Grb10
,
which could alter its self-association properties, although the BPS and
SH2 domains are identical in all Grb10 isoforms.
F1) in the dimer interface.
B9 (Tyr-516 in
Grb10), adjacent to Phe-515 in the center of the dimer interface (Fig.
1), is a histidine in Grb7, which might result in a weakened
dimerization interface for the Grb7 SH2 domain.
subunits of the insulin
and IGF1 receptors. Interestingly, in the structures of the
phosphorylated tyrosine kinase domains of these receptors (32, 46), the
activation loop makes an abrupt turn after the first phosphotyrosine
pTyr-1158/pTyr-1131 (insulin/IGF1 receptor). This conformation of the
phosphorylated activation loop, including the turn after
pTyr-1158/pTyr-1131, is stabilized by numerous interactions.
D6, which
is a leucine in Grb7 and a glutamine in Grb10/Grb14. Based on the Grb10
SH2 domain structure (with superimposed SH2-phosphopeptide structures),
a leucine at
D6 can make favorable hydrophobic contacts with the phenolic ring of the phosphotyrosine. It should be noted that the
D6
residue is variable in SH2 domains and is often not a hydrophobic residue.
D6 (14),
arginine at BC2 (predicted), and possibly others "rescue" the
ability of Grb7 to bind phosphopeptides with reasonable affinity.
and the insulin receptor, as depicted in
Fig. 6B. The SH2 dimer binds to the phosphorylated kinase
activation loops in the two insulin receptor
subunits, with
pTyr-1158 in the canonical phosphotyrosine-binding pocket of each SH2
domain. Yeast two-hybrid experiments demonstrate that pTyr-1162 and/or pTyr-1163 are critical for the interaction between the Grb10 SH2 domain
and the insulin receptor, with pTyr-1158 less important (4, 11). The
dependence on pTyr-1162/1163 is probably due in part to the role of
these phosphotyrosines, especially pTyr-1163, in stabilization of the
activation loop conformation (32). The aberrant BC loop (involved in
phosphate coordination) in the Grb10 SH2 domain could explain why the
substitution Tyr-1158
Phe leads to only a modest decrease in
binding of the SH2 domain, assuming additional interactions exist
between the SH2 domain and IRK. The PH domain of Grb10
is predicted
to facilitate localization of Grb10
to the plasma membrane, which
will promote SH2 domain dimerization.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Till and S. Li for assistance in synchrotron data collection and helpful discussions and N. Covino for technical support.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK52916 (to S. R. H.). Financial support for beamline X12C of the National Synchrotron Light Source comes principally from the National Institutes of Health and the Department of Energy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1NRV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence should be addressed: Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Ave., New York, NY 10016. Tel.: 212-263-8938; Fax: 212-263-8951; E-mail: hubbard@saturn.med.nyu.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212026200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PH, pleckstrin homology; SH2, Src homology 2; BPS, between PH and SH2; IGF, insulin-like growth factor; P+3, third residue after phosphotyrosine; WT, wild type; IRK, insulin receptor kinase domain; LCK, lymphocyte kinase.
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