From the Program in Molecular Medicine, University of
Massachusetts Medical School, Worcester, Massachusetts 01605 and the
¶ Department of Medicine, Vanderbilt University,
Nashville, Tennessee 37232
Received for publication, October 11, 2002, and in revised form, December 4, 2002
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ABSTRACT |
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SHP-1 is a cytosolic protein-tyrosine phosphatase
that behaves as a negative regulator in eukaryotic cellular signaling
pathways. To understand its regulatory mechanism, we have determined
the crystal structure of the C-terminal truncated human SHP-1 in the inactive conformation at 2.8-Å resolution and refined the structure to
a crystallographic R-factor of 24.0%. The
three-dimensional structure shows that the ligand-free SHP-1 has an
auto-inhibited conformation. Its N-SH2 domain blocks the catalytic
domain and keeps the enzyme in the inactive conformation, which
supports that the phosphatase activity of SHP-1 is primarily regulated by the N-SH2 domain. In addition, the C-SH2 domain of SHP-1 has a
different orientation from and is more flexible than that of SHP-2,
which enables us to propose an enzymatic activation mechanism in which
the C-SH2 domains of SHPs could be involved in searching for
phosphotyrosine activators.
Tyrosine phosphorylation is a key mechanism for regulating
eukaryotic cellular signaling pathways. The protein tyrosine
phosphorylation level is precisely regulated by two types of enzymes:
protein-tyrosine kinases
(PTKs)1 and protein-tyrosine
phosphatases (PTPs), in which PTPs act to counter-balance the process
through dephosphorylation of the phosphorylated tyrosines (1, 2). PTPs
can be divided into two groups, receptor protein-tyrosine phosphatases
and cytosolic protein-tyrosine phosphatases. The SH2 domain-containing
PTPs, SHP-1 and SHP-2, are both cytosolic PTPs and share many
structural and regulatory features. They both have two tandem SH2
domains at the N terminus followed by a single catalytic domain and an
inhibitory C-terminal tail. However, irrespective of similar structural
and regulatory characteristics, these two enzymes have different
biological function in vivo.
Different from SHP-2, which is expressed in all kinds of tissues, SHP-1
is predominantly expressed in hematopoietic and epithelial cells and
behaves mainly as a negative regulator of signaling pathways in
lymphocytes (1, 2). SHP-1 is dormant in the cytosol, with its
phosphatase activity inhibited by both the SH2 domains and the
C-terminal tail (1, 3-5). In response to an activation signal, SHP-1
is recruited to membrane-bound inhibitory receptors via the binding of
its SH2 domains to the tyrosine-phosphorylated immunoreceptor
tyrosine-based inhibitory motif within the cytoplasmic domain of a
receptor (6-8). During this process, SHP-1 undergoes a structural
rearrangement, exposes its active site, and binds to the downstream
substrates, thereby dephosphorylating the substrates to turn off the
cellular signals.
SHP-1 also presents in several types of non-hematopoietic cells
(9-12). Overexpression of a catalytically inactive SHP-1 mutant in
these cells strongly suppressed mitogen-activated pathways, reducing
signal transduction and activation of transcription; these findings
demonstrate that SHP-1 has a positive effect on mitogenic signaling in
these non-hematopoietic cells (10, 11). Thus, SHP-1 probably has both
the negative and positive regulatory function in different types of
cells. Many aspects of the molecular mechanism that underlies the
activation and regulation of SHP-1 remain unclear.
Here, we report the 2.8-Å crystal structure of the 61-residue
C-terminal-truncated SHP-1 (residues 1-532) in the ligand-free dormant
conformation. SHP-1 shares a 60% overall sequence identity with SHP-2.
A comparison of the crystal structures of SHP-1 and SHP-2 reveals that
the C-SH2 domain of SHPs is highly mobile, suggesting that this domain
may be involved in searching for phosphopeptides to activate the enzyme
in the initial stages of enzyme activation.
Protein Expression, Purification, and Crystallization--
The
61-residue C-terminal-truncated SHP-1 (residues 1-532) was cloned and
expressed as described previously (13), except that the
Escherichia coli strain of BL21 (pLysS) was used for the
expression to increase the protein solubility. Purification was
operated by ion exchange and affinity chromatography. The harvested
protein sample was desalted by centrifugation and concentrated to 3 mg
ml Data Collection and Structural Determination--
A 2.8-Å
diffraction data set was collected at 100 K on the F1 beamline at
CHESS (Cornell University). The x-ray wavelength was 0.978 Å. 15%
glycerol was introduced into the reservoir solution as cryoprotectant
to reduce the freezing damage to crystals. Data were processed with
DENZO and SCALEPACK (14). The space group of the SHP-1 crystals belongs
to P212121, and the unit-cell
parameters were determined to be a = 44.74 Å,
b = 100.40 Å, and c = 149.47 Å. There
was one molecule per asymmetric unit.
The structure of SHP-1 was solved by the molecular replacement method
with AmoRe (15), using the coordinates of C-terminal truncated SHP-2
(16) as the search model. Refinement was performed with CNS (17). The
refinement process was monitored with the free R-factor that
was calculated using randomly selected 10% of the data as the test
set. After the initial rigid-body refinement, a 2Fo Overall Structure of SHP-1--
The crystallized ligand-free human
protein-tyrosine phosphatase SHP-1 contains residues 1-532 but lacks
the 61-residue C-terminal tail. Its crystal structure has been
determined by molecular replacement method and refined to a final
crystallographic R-factor of 24.0% at 2.8-Å resolution.
The statistics for the crystal data and refinement results are shown in
Table I.
Three domains were defined well from the present crystal structure. The
residues 1-108 and 116-208 fold as two Src homology 2 domains, the
N-SH2 and C-SH2 domains, respectively. Residues 270-532 fold as the
typical PTP domain, a highly twisted ten-stranded Comparison of the SHP-1 Structure with the Domain-alone
Structures--
SH2 domain is responsible for recognizing the
phosphotyrosine-containing proteins, thereby facilitating
phosphorylation-dependent protein-protein interactions that
result in signal propagation (19). In general, residue at the
(p + 1) position of the phosphotyrosine peptide of the
binding target is considered most important for defining the binding
specificity (20). Crystal structure revealed that both SH2 domains of
SHP-1 have the typical SH2 domain fold, which consists of a central
four-stranded
The structure of the PTP domain of ligand-free SHP-1 is almost
identical to that of the peptide-bound catalytic domain of SHP-1 (Fig.
3B). Superimposition of the Auto-inhibited Structure of SHP-1--
Previous studies have
revealed that both SH2 domains of SHP-1 could bind to
tyrosine-phosphorylated immunoreceptor tyrosine-based inhibitory motif
peptides (8). Similar to the crystallographic data for its close
relative SHP-2, however, the crystal structure of the ligand-free SHP-1
supports that the N-SH2 domain, instead of C-SH2 or both domains, acts
the auto-inhibition role. The interaction between N-SH2 domain and PTP
domain is extensive, whereas the C-SH2 domain does not show significant
interface with either of the other two domains.
Cys455, the catalytic nucleophile, is located at the base
of the active-site cleft. In the auto-inhibited conformation of SHP-1, it appears that the N
In addition, the phosphopeptide-binding sites of both SH2 domains in
SHP-1, like in SHP-2, appear to expose to the solvent but not to the
active site, which implies that SHP-1 repression by the N-SH2 domain is
most likely to involve conformationally mediated inhibition of
substrate binding (16). Molecular dynamics simulation for SHP-2 also
identified that the conformational flexibility of the C-terminal half
of the N-SH2 domain that interacts with the PTP domain was
significantly larger in the absence of phosphopeptide ligand than that
in the ligand-complexed N-SH2 domain (24). Homology in both primary and
three-dimensional structures of SHP-1 and SHP-2 suggests a similar
mechanism of the interaction between the N-SH2 and PTP domains.
Structural Comparison with SHP-2--
Determination of the crystal
structure of SHP-1 revealed that its three domains are similarly
assembled to those in SHP-2 (16). High structural similarity was
observed between SHP-1 and SHP-2, especially in the PTP and N-SH2
domains (Fig. 3, B and C). Superimposition of the
N-SH2 domains of SHP-1 and SHP-2 produced an r.m.s. deviation of only
1.3 Å for their C
However, the C-SH2 domain in SHP-1 structure is not only differently
orientated from that in SHP-2, but its secondary structure elements are
relatively more openly organized. The additional superimposition of the
C-SH2 domains of SHP-1 and SHP-2, following the superimposition of
their N-SH2 and PTP domains (Fig. 3A), revealed an up to
56.3-degree rotation of their orientations and a 17.1-Å translation of
their mass centroids (Fig. 3D). Consequently, the
phosphotyrosine-binding pocket (27) of the C-SH2 domain undergoes wide
ranging rotation.
The conformation of the N-SH2 domain in SHP-1 appears changed compared
with the complex structure of the phosphotyrosine peptide-bound N-SH2
domain of SHP-2. As indicated in Fig. 3C, both the
N Activation Mechanism of SHP-1--
The presence of multiple SH2
domains leads to a problem of the selectivity on phosphopeptide ligands
(19). Earlier studies established that N-SH2 domain in SHPs plays more
critical role than C-SH2 domain in signaling; however, the C-SH2 domain
is indispensable for optimal signaling (2). Although structural
comparison of the C-SH2 domains of our ligand-free structure with the
peptide-bound C-SH2 domain of SHP-2 provided no direct information
about its function, the observed structural differences may suggest
that the C-SH2 domain is responsible for searching for the enzyme
activator. Together with the special assembly of these three domains, a
model was proposed the activation mechanism for SHP-1 (Fig.
4). In this model, the highly mobile
C-SH2 domain functions as an antenna to search for phosphopeptides.
Binding of phosphopeptide to the C-SH2 domain results in large
conformational changes that restore the distorted conformation of the
neighboring N-SH2 domain and subsequently opens up its
phosphopeptide-binding pocket to harbor a second phosphopeptide
molecule. These events can weaken the auto-inhibiting interaction on
the interface between the N-SH2 and PTP domains and permit the
subsequent synergistic opening up of the active site of the PTP domain.
This mechanism is consistent with the notion that a truncated SHP-1
lacking the C-SH2 domain would be activated to a much lesser extent
than full-length SHP-1 (25, 26). It also is envisioned that a much
larger change in the relative positions of the two SH2 domains will
occur due to the mobility of the C-SH2 domain when they are
simultaneously bound by biphosphorylated peptides, which can lead
to greater movement of the N-SH2 domain. This movement should optimize
the opening up of the active site of the PTP domain and can
qualitatively explain why biphosphorylated peptides can activate SHPs
at a 10-fold higher level than monophosphorylated peptides (28).
Although we have already had the basic concepts of the differentiation
and cooperation of these three domains based on the crystallographic
and biochemical data, many questions still remain unclear for SHPs.
Sequence analysis and structural comparison have revealed highly
similar primary and three-dimensional structures of these two
C-terminal truncated enzymes. In addition, the C-terminal truncated
SHP-1 and the C-terminal truncated SHP-2 could both be activated in an
identical manner to their corresponding wild-type enzymes.
Nevertheless, although both have the similar phosphorylation sites, the
C-terminal tails (~66 residues) of the full-length SHPs share very
low sequence homology (<15%), in contrast to the overall high
homology (60%). Biochemical and immunological studies showed that the
tails are associated with the lipid-binding and subcellular
localization of these enzymes (29-30). Therefore, further crystallographic and biochemical studies will be required to understand how the tails regulate the functions of these enzymes and how to
cooperate with the other three domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 for crystallization. Crystallization was performed by
the vapor-diffusion hanging-drop method at 4 °C. The crystallization
drops composed of equal-volume mixture (3 µl:3 µl) of protein
solution and reservoir solution (25-30% polyethylene glycol 8000, 5 mM dithiothreitol, 0.1 M HEPES, pH 7.0)
were equilibrated against the reservoir solution (0.5 ml), and crystals
were obtained within 2 weeks.
Fc electron density map was calculated. The C-SH2
domain was found to apparently mismatch the first
2Fo
Fc map. Therefore, the
C-SH2 domain was omitted from the model. Thereafter, five cycles of
positional refinement and re-building the model for the N-SH2 and PTP
domains were applied for all data in the resolution range of 10-3.0
Å, and an Fo
Fc map was
generated. Subsequently, the C-SH2 domain was manually re-assigned
based on the omitted Fo
Fc map. The full model was refined with the 2-
cutoff data in the 10-2.8-Å resolution range. The model building interspersed with CNS
refinement was done with TURBO-FRODO (18). In the penultimate cycle,
the free R-factor was reduced to 33.4%, correspondingly, the final crystallographic R-factor was 24.0%, with all
reflections used in the refinement. In the final model, the linker
regions between the C-SH2 domain and the N-SH2 and catalytic domains
were not observed. No water molecules were assigned to the final model. The crystal statistics, final refinement results, and geometric analyses are summarized in Table I.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Data collection and refinement characteristics for SHP-1
-sheet flanked by
four helices on the convex side and two helices and a
-hairpin from
the concave side. The N-terminal extended region of the PTP domain
comprising helices
0 and
1' are defined well, whereas the linker
regions of C-SH2 to N-SH2 and to PTP domains, residues 109-115 and
209-231, were disordered and not observed on the finial electron
density map. The architecture of the three domains is compact. The two
SH2 domains look like two antennas of the global PTP domain in the
overall view. Fig. 1 shows a ribbon
representation of the final model (Fig. 1A), the
intramolecular interaction at the interface between the N-SH2 and PTP
domains (Fig. 1B), and a portion of an electron density map
of the flexible C-SH2 domain (Fig. 1C). Fig.
2 shows the sequence alignment of SHP-1
with three of its homologues, with the secondary structure elements
assigned.
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Fig. 1.
Overall structure of SHP-1. A
schematic drawing of the structure with the color ramping from
blue (N-terminal) to red (C-terminal) is shown
(A). The secondary structure elements are labeled according
to the domains to which they belong. The dashed lines
indicate disordered regions. The interactions between the N 4-N
5
hairpin loop of the N-SH2 domain and the active site of the PTP domain
are shown by the accessible surface for the PTP domain and bonding
models for the interactions (B). A stereo view of the
2Fo
Fc electron density map
around the C-SH2 domain was contoured at 1.0
(C). These
figures were prepared with MOLSCRIPT (31), RASTER3D (32), and
GRASP (33).
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Fig. 2.
Sequence alignment and secondary structure
element assignments for SHPs and their homologues.
Identical and similar residues are cyan and
green, respectively. Secondary structure elements for SHP-1
and SHP-2 are shown in blue and orange,
respectively.
-sheet with an
-helix on either side. The
phosphopeptide-binding sites of both SH2 domains face away from the PTP
domain and are fully exposed on the surface of the molecule (Fig.
3A). Nevertheless, the spatial
arrangement of the two SH2 domains on PTP domain significantly differs.
In contrast to the N-SH2 domain that strongly interacts with the PTP
domain (see below), the C-SH2 domain is tethered around and extends to
the surface of the catalytic domain and has no significant direct
interactions with the PTP domain.
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Fig. 3.
Structural comparison of SHP-1 and its
domains with SHP-2 (A), the PTP domain
(B) complexed with Tyr(P)469 (yellow
coil) (21), and the N- and C-terminal SH2 domains (27)
(C and D) of SHP-2. All
superimposition of the structures of SHP-1 and SHP-2 is based on the
C atoms. The phosphopeptide-binding sites of the two SH2 domains and
the active site of the PTP domain are indicated by the
yellow and red dashed ellipses, respectively
(A). The loop regions with large conformational changes are
labeled, in which the conformational change of
5-
6 hairpin is
highlighted in the green box (B). A part of the
accessible surface is shown for the C-SH2 and PTP domains of SHP-1, and
the loop regions with large conformational changes are labeled
(C).
-carbons between the structure of the auto-inhibited PTP domain and the previously reported
structure of the catalytic domain of SHP-1 complexed with
Tyr(P)469-peptide (21) gives a root mean square
(r.m.s.) deviation of 1.9 Å. Nevertheless, the conformations of the
5-
6 hairpin loops (residues 356-363) in these two structures
significantly differ. In the present structure, the C
of
Gly359 in the
5-
6 loop is shifted 6.7 Å, reflecting
an ~25-degree rotation of the hairpin's "plane" (Fig.
3B). It could be expected that the flexibility of this loop
region was related to its important role in substrate recognition
(21-23). Superimposition of these structures also shows flexibility
around the phosphopeptide-binding sites of some other loops, with
conformational changes of 1.5-2.0 Å for the
1-
1 and
3-
2
loops (Fig. 3B). In addition, the
0 helix appears to be
highly mobile. In the Tyr(P)469-PTP complex structure (21),
the
0 is located at the N terminus, far away from the PTP domain. In
the present SHP-1 structure, the
0 helix bridges the C-SH2 and PTP
domains and rotates ~60 degrees to approach the surface of the PTP
domain (Fig. 3B). This mobility reflects the high
flexibility of the
0-
1 loop, which may be required for exposure
of the active site during enzyme activation. Our previous research also
lends support to the cooperative role of helix
0 in the substrate
recognition (23).
4-N
5 loop of the N-SH2 domain is protruding to the catalytic PTP domain to directly block the entrance to the
active site, which prevents the cysteine residue from exposing to the
substrate (Figs. 1B and 3C). This inactive
conformation is stabilized by various interactions including the salt
bridge between Asp61 and Lys362, along with the
-
interaction between the Phe62 and
Tyr278 side chains, and the hydrogen bonds in the residue
pairs of Ser59/His422,
Gly60/Gln506, and
Asn58/Gln502. All of these amino acid residues
involved in the interactions are conserved well between SHP-1 and SHP-2
except for Ser59; SHP-2 has a threonine residue at the corresponding
site (Fig. 2). Likewise, most of these residues are also highly
conserved in both CSW and PTP2 (Fig. 2), implying the similar
interdomain interactions in these two enzymes. In addition, the
extensive interactions around the protruding N
4-N
5 loop are
present at the interface between the N-SH2 domain and the PTP domain
(Fig. 3C). The area of interacting interface between these
two domains is calculated to be 1478.9 Å2, which is 20%
larger than that in SHP-2.
atoms. The interactions between the N-SH2 and PTP
domains defined from the structure support the previously proposed
regulatory mechanism that both SHP-1 and SHP-2 use the N-SH2 domain to
keep the enzyme in the inactive conformation (2, 25, 26).
2-N
7 and N
7-N
8 loops of N-SH2 domain in SHP-1 project
about 5 Å toward the phosphotyrosine-binding pocket and block the
binding site for the phosphotyrosine peptide (27). This indicates that
stronger interactions exist between the N-SH2 and PTP domains prior to the binding of phosphotyrosine peptide to the N-SH2 domain.
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Fig. 4.
A proposed mechanism for the activation of
SHP-1. From left to right, the three
structural models represent the auto-inhibited, C-SH2
domain-stimulated, and activated conformations. The molecular surface
of the PTP domain is shown in orange. The SH2 domains
modeled from SHP-1 and SHP-2 structures are depicted by
blue and red coils, respectively. This figure was
prepared with SETOR (34).
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FOOTNOTES |
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* This work was supported by a Career Development Award from the American Diabetes Association (to G. W. Z.), the Pilot project from Diabetes and Endocrinology Research Center program of the University of Massachusetts Medical School (to G. W. Z.), and by National Institutes of Health Grants AL45858 (to G. W. Z.) and HL57393 and CA75218 (both to Z. J. Z.).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.
§ Current address: Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South St., Waltham, MA 02454.
To whom correspondence should be addressed: Program in
Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Worcester, MA 01605. Tel.: 508-856-6869; Fax: 508-856-1218; E-mail: wayne.zhou@umassmed.edu.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M210430200
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ABBREVIATIONS |
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The abbreviations used are: PTK, protein-tyrosine kinase; PTP, protein-tyrosine phosphatase; SH2, src homology 2; r.m.s., root mean square; CSW, PTP2.
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