From the Molecular Medicine Laboratory and Macromolecular Crystallography Unit, Division of Experimental Medicine, Harvard Institutes of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 9, 2002, and in revised form, November 14, 2002
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ABSTRACT |
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Erbin contains a class I PDZ domain that binds to
the C-terminal region of the receptor tyrosine kinase ErbB2, a class II ligand. The crystal structure of the human Erbin PDZ bound to the
peptide EYLGLDVPV corresponding to the C-terminal residues 1247-1255 of human ErbB2 has been determined at 1.25-Å resolution. The Erbin PDZ deviates from the canonical PDZ fold in that it contains
a single PDZ1 (PSD-95/DLG/ZO-1)
domains are protein interaction modules that play fundamental roles in
the assembly of membrane receptors, ion channels, and other molecules
into signal transduction complexes known as transducisomes (1-3). The
PDZ fold comprises a six-stranded antiparallel Erbin was originally identified as a protein that interacts with the
receptor tyrosine kinase ErbB2 (also known as HER-2 or Neu) and plays a
role in its localization at the basolateral membrane of epithelial
cells (8, 9). Recent studies have shown that Erbin is also highly
concentrated at neuronal postsynaptic membranes and neuromuscular
junctions, where it interacts with ErbB2 (10). Erbin contains a class I
PDZ domain that binds with high affinity to the sequence DSWV present
at the C termini of Here, we present the crystal structure of the Erbin PDZ bound to the
ErbB2 C terminus. The structure reveals a novel interaction of the
peptidic Tyr Protein Crystallization--
A DNA fragment encoding the human
Erbin PDZ domain (residues 1280-1371) was amplified from Quick-Clone
cDNA (Clontech) using the polymerase chain
reaction and cloned into a modified pGEX-2T vector. The Erbin PDZ was
expressed in Escherichia coli BL21(DE3) cells as a
glutathione S-transferase fusion, purified on
glutathione-Sepharose 4B, released with thrombin digestion, and further
purified by gel filtration (5). The Erbin PDZ protein (19 mg/ml in 500 mM NaCl, 50 mM Tris-HCl, pH 8.3) was mixed with
the synthetic peptide EYLGLDVPV at an equimolar ratio and crystallized
in 12-15% polyethylene glycol 4000, 10% glycerol, 100 mM
ammonium acetate, 100 mM sodium acetate, pH 4.6, at
20 °C, using the sitting drop vapor diffusion method. Crystals were
cryoprotected in mother liquor containing 30% glycerol and
flash-frozen in a liquid nitrogen stream. The mutation V1366M was
introduced in the Erbin PDZ using the polymerase chain reaction, and
the resulting protein was expressed in B834(DE3)pLysS cells grown in
minimal medium supplemented with 40 mg/l selenomethionine
(SeMet). The SeMet-protein was purified and co-crystallized with the
ErbB2 peptide under similar conditions. Multiwavelength anomalous
dispersion (MAD) data sets of the SeMet-substituted PDZ(V1366M)-peptide
crystals were collected at 100 K using synchrotron radiation at the
Cornell High Energy Synchrotron Source (F2 station), Ithaca, NY. High
resolution data of isomorphous crystals of the wild-type Erbin
PDZ-peptide complex were also collected at the F2 station. The crystals
belong to space group P21 with unit cell dimensions
a = 26.6 Å, b = 57.4 Å,
c = 30.4 Å, Structure Determination and Refinement--
The crystal
structure of the SeMet-substituted PDZ(V1366M)-peptide complex was
determined using SOLVE/RESOLVE (19). The obtained phases were used to
solve the structure of the wild-type Erbin PDZ-peptide complex at
1.25-Å resolution. Phase extension and automated model building were
performed using wARP (20), in combination with manual intervention
using O (21). Initial isotropic refinement was performed using REFMAC
(22), followed by several rounds of anisotropic refinement with
SHELXL-97 (23). The structure of the Erbin PDZ-phosphopeptide complex
was determined by molecular replacement with AMoRe (24) using the Erbin
PDZ as the search model. The crystallized PDZ domain includes the vector-derived residues GSM at its N terminus. In the 1.25-Å structure the side chains of PDZ residues Glu1280,
Ser1294, Ser1325, His1347,
Gln1349, and Ile1365 are modeled in two
conformations. The main conformation of His1347 (occupancy
0.7) has excellent electron density and is used to describe the present
structure, whereas the electron density for the minor conformation is
of poor quality.
Isothermal Titration Calorimetry--
Binding constants of the
Erbin PDZ to the ErbB2 peptides were measured using a VP-ITC
microcalorimeter (MicroCal, LLC). Briefly, a 0.896 mM
solution of the native and a 0.830 mM solution of
the phosphotyrosine-containing ErbB2 peptide were titrated into a 0.0389 mM solution of Erbin PDZ protein in 25 mM Tris-HCl, pH 8.3, at 25 °C. Titration curves were
analyzed using the program ORIGIN 5.0 (OriginLab).
Structural Basis for Class II Ligand Recognition by the Erbin
PDZ--
The crystal structure of the Erbin PDZ bound to the ErbB2
peptide EYLGLDVPV was determined using MAD phasing and was refined anisotropically to 1.25-Å resolution. The Erbin PDZ lacks the short
The ErbB2 peptide inserts into the Erbin PDZ ligand-binding groove
antiparallel to the The Phosphorylation of Tyr
Superposition of the Erbin PDZ structures with the PSD-95 PDZ3 (4)
reveals that Val 0, Pro Structural and Functional Implications--
The property of the
newly discovered pocket P2 to discriminate between the
phosphorylation states of Tyr
Do other PDZ domains have a P2 pocket? In contrast to the
short -helix. The isopropyl group of valine at position
2 of
the ErbB2 peptide interacts with the Erbin Val1351
and displaces the peptide backbone away from the
-helix, elucidating the molecular basis of class II ligand recognition by a class I PDZ
domain. Strikingly, the phenolic ring of tyrosine
7 enters into a
pocket formed by the extended
2-
3 loop of the Erbin PDZ. Phosphorylation of tyrosine
7 abolishes this interaction but does not
affect the binding of the four C-terminal peptidic residues to PDZ, as
revealed by the crystal structure of the Erbin PDZ complexed with a
phosphotyrosine-containing ErbB2 peptide. Since phosphorylation of
tyrosine
7 plays a critical role in ErbB2 function, the selective
binding and sequestration of this residue in its unphosphorylated state
by the Erbin PDZ provides a novel mechanism for regulation of the
ErbB2-mediated signaling and oncogenicity.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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-barrel capped by two
-helices (1-6). PDZ domains interact with C-terminal peptides and
are currently classified into two major categories based on their
target sequence specificity. Class I domains bind to peptides with the
consensus X-(S/T)-X-
(X denoting
any amino acid and
representing a hydrophobic residue), whereas
class II domains recognize the motif X-
-X-
(1-3). The residues at positions 0 and
2 of the peptide (position 0 referring to the C-terminal residue) play a critical role in the
specificity and affinity of the interaction, whereas it is believed
that amino acids upstream of the
5 position do not interact with PDZ
(1-7). However, the structural determinants of ligand selectivity by PDZ domains are more complex than initially thought. For example, recent studies established an important contribution of the penultimate peptidic residue in the PDZ-ligand interaction (5, 6). Furthermore, several PDZ domains have sequence specificities that do not fall into
the two classes implying the existence of more categories, whereas
others bind both class I and II ligands, suggesting an intrinsic
flexibility in these modules to accommodate both polar and non-polar
side chains at position
2 (1-3).
-catenin, ARVCF, and p0071 (11, 12). Notably,
the ErbB2 sequence EYLGLDVPV that is recognized by the Erbin PDZ (8,
13), is a class II ligand, posing an interesting structural problem
regarding the molecular mechanisms underlying the dual ligand
specificity of this domain. The Erbin PDZ binds preferentially to the
ErbB2 tail having an unphosphorylated tyrosine at position
7
(corresponding to Tyr1248 in full-length human ErbB2),
whereas phosphorylation of this residue reduces significantly the
affinity of the Erbin-ErbB2 interaction (8). This preference for an
unphosphorylated tyrosine is intriguing, because a PDZ interaction with
the peptidic residue
7 has not been observed in previous structural
studies (1-7). Importantly, phosphorylation of Tyr1248
following ErbB2 activation is a critical event for the mitogenic signaling and oncogenicity of this receptor (14-16). Moreover, Tyr1248 plays an important role in the basolateral
localization of ErbB2 (17).
7 with the extended
2-
3 loop of the Erbin PDZ. A
second crystal structure of this domain bound to a
phosphotyrosine-containing ErbB2 peptide shows that phosphorylation of
Tyr
7 abrogates its interaction with the
2-
3 loop. These
results suggest new mechanisms for regulation of the ErbB2-mediated
signaling through its dynamic interaction with the Erbin PDZ.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
= 100.6°. Crystals of the Erbin
PDZ bound to the peptide EpYLGLDVPV (pY denoting phosphotyrosine) were
obtained under similar conditions and were analyzed at 100 K using
CuK
radiation. The crystals belong to space group P21
with a = 26.5 Å, b = 57.0 Å,
c = 30.9 Å,
= 99.2°. Data were processed
using DENZO and SCALEPACK (18) (Table
I).
Statistics of structure determination and refinement
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix that is present between the
3 and
4 strands in PDZs
with known structure (1-7), due to the shorter length of the Erbin
3-
4 loop (Fig. 1, A and
B). The significance of this deviation from the canonical
PDZ fold is unclear because this
-helix has no known function (1-3)
and its inconsequential absence from the Erbin PDZ argues against a
structural role in the folding of this module.
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Fig. 1.
Structure of the Erbin PDZ bound to the
unphosphorylated ErbB2 peptide. A, sequence
comparison of selected class I PDZ domains. Identical residues in four
or more domains are shown as white letters on
blue background. Hyphens represent gaps inserted
for optimum alignment. The secondary structure of the Erbin PDZ is
indicated at the top. Residues forming a short -helix in
PDZs with known structures are enclosed in a red box.
B, stereo view of the Erbin PDZ bound to the peptide
EYLGLDVPV. The figure was made using BOBSCRIPT (30) and POV-Ray
(www.povray.org). C, surface topology of the Erbin PDZ bound
to the ErbB2 peptide. The figure was made using GRASP (31).
D, two-dimensional representation of the interactions
between Erbin PDZ residues (orange) and the peptide
(purple). Water molecules (W) are shown as
cyan spheres, hydrogen bonds as dashed lines, and
hydrophobic interactions as arcs with radial
spokes. The figure was made using LIGPLOT (32). E,
stereo view of a weighted 2Fobs
Fcalc electron density map at the P2
pocket calculated at 1.25 Å and contoured at 2.5
.
2 strand, extending and twisting the
-sheet
of PDZ (Fig. 1, B and C). The isopropyl and
carboxylate groups of Val 0 enter into the carboxylate-binding pocket
(designated here as P1), where they are stabilized through
hydrophobic interactions and hydrogen bonds with PDZ residues (Fig.
1D), similar to those described for other class I PDZ-ligand
complexes (1-6). Remarkably, the isopropyl group of Val
2 makes
hydrophobic contacts with Val1351, which appear to cause a
displacement of the peptide backbone away from the
-helix (Fig.
1B), providing an explanation for the ability of Erbin PDZ
to recognize a class II ligand. The peptide is further stabilized at
this position through an interaction of Asp
3 with
Thr1316 (Fig. 1D), whereas Leu
4, Gly
5, and
Leu
6 do not bind to PDZ. Interestingly, the imidazole ring of the
conserved His1347, which is the hallmark of class I PDZ
domains and plays a critical role in the selection of the residue
2,
points away from Val
2, where it hydrogen bonds with the carbonyl
oxygen of Gly1299 (Fig. 1B).
2-
3 Loop of Erbin PDZ Interacts with Tyr
7 of the ErbB2
Ligand--
Strikingly, the phenolic ring of Tyr
7 folds back in a
direction parallel to the peptidic backbone and enters a pocket,
designated P2, which is formed by Ser1296 in
the
2 strand and Gly1303, Asn1304, and
Pro1305 in the
2-
3 loop (Fig. 1, B and
C). This represents the first structural evidence for a
direct interaction of the PDZ domain with the peptidic residue
7. The
2-
3 loop of Erbin PDZ is considerably longer than that of PDZs
with known structure (Fig. 1A) and contains five glycine and
two proline residues that create a bent platform against which Tyr
7
is stacked. The phenolic ring of Tyr
7 is stabilized mainly by
hydrophobic interactions and is well ordered, as indicated by the high
quality electron density map (Fig. 1E). The hydroxyl group
of Tyr
7 hydrogen bonds through two ordered water molecules with Asp
3 (Fig. 1D).
7 Abolishes Binding to the P2
Pocket--
Because phosphorylation of Tyr1248 plays a
critical role in ErbB2 signaling (14-16), we also determined the
crystal structure of the Erbin PDZ bound to the peptide EpYLGLDVPV. No
electron density is observed for the peptidic residues
5 to
8 and
the P2 pocket is empty (Fig.
2A). In contrast, Val 0, Pro
1, Val
2, Asp
3, and Leu
4 are well ordered inside the
ligand-binding groove (Fig. 2A). The integrity of the
peptide in the crystallized complex was verified by mass spectroscopic
analysis (data not shown), indicating that the invisible portion of the
peptide is disordered and faces toward the solution. Isothermal
titration calorimetry experiments showed that the native ErbB2 peptide
binds to the Erbin PDZ with a Kd of ~50
µM, whereas the phosphotyrosine-containing peptide binds
to PDZ with a Kd of ~128 µM. The
~2.5-fold reduction in the affinity of Erbin PDZ for the
phosphorylated ErbB2 peptide is attributed to the loss of the
hydrophobic interactions and hydrogen bonds stabilizing the phenolic
ring of Tyr
7 inside the P2 pocket.
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Fig. 2.
Structure of the Erbin PDZ bound to the
phosphorylated ErbB2 peptide. A, stereo view of
the Erbin PDZ bound to the peptide EpYLGLDVPV. A weighted
2Fobs Fcalc electron
density map calculated at 1.88-Å resolution and contoured at 1.0
is superimposed on the ErbB2 peptide. B, superposition of
the C
backbone traces of Erbin PDZ-peptide (pink), Erbin
PDZ-phosphopeptide (blue), and PSD-95 PDZ3-peptide
(yellow) (Protein Data Bank code 1BE9). Side chains of the
peptidic residues, Erbin His1347 and Val1351,
and PSD-95 His372 are shown as stick
models.
1, Val
2, and Asp
3 are superposed extremely well in both Erbin complexes, whereas the ErbB2 backbone is
displaced away from the
-helix as compared with PSD-95 PDZ3 (Fig.
2B). These results indicate that the displacement of the ErbB2 peptide is due to the Val
2 interaction with
Val1351 rather than the Tyr
7 binding to P2.
Only small differences are observed in the backbone positions of the
Erbin
2-
3 loops (overall root-mean-square deviation 0.26 Å for
residues 1299-1311), indicating that the P2 site is
preformed and does not undergo major conformational changes upon Tyr
7 binding. By contrast, the
2-
3 loops of the Erbin PDZ and
PSD-95 PDZ3 occupy completely different positions and are not superimposable.
7 indicates that it may play a
regulatory role in ErbB2 signaling and suggests an attractive model for
this regulation. Conceivably, during the inactive state of ErbB2, Tyr
7 is buried inside P2 and is inaccessible for
phosphorylation and interaction with other proteins. Activation of
ErbB2 triggers the release of Tyr
7 from P2, possibly
through conformational changes induced in Erbin and/or the cytoplasmic domain of ErbB2. Notably, Erbin becomes phosphorylated by ErbB2 following receptor activation (8), raising the intriguing possibility that this may represent a step preceding the dissociation of Tyr
7.
Subsequently, the released tyrosine is primed for phosphorylation and
interaction with phosphotyrosine-binding domains (e.g. PTB or SH2) of downstream signaling proteins (14, 15). Following signal
transduction, dephosphorylation of Tyr
7 restores its original
position inside P2. Importantly, in contrast to the
regulatory site P2 that oscillates between bound and
unbound states, P1 interacts constitutively with the last
four residues of ErbB2 securing the continuous participation of Erbin
and ErbB2 in the same macromolecular complex at the basolateral
membrane throughout the activation-inactivation cycles of the receptor.
This model also allows for simultaneous binding of the Erbin PDZ and
either PTB or SH2 domains to the phosphorylated ErbB2 C-terminal
region, because these modules have non-overlapping recognition motifs.
2-
3 loops of PSD-95 PDZ3 and NHERF PDZ1 (Fig.
1A) that have not been shown to interact with peptidic
residues (4-6), the extended
2-
3 loops of the PSD-95 PDZ1,
PSD-95 PDZ2, and PTP1E PDZ2 domains are involved in ligand interactions
(7, 25-28). Importantly, alternative spliced isoforms of PTP1E PDZ2
with different
2-
3 loop lengths have entirely different binding
affinities for the C-terminal region of the tumor suppressor protein
APC (29), providing further evidence for an important role of
P2 in PDZ-ligand interactions. These observations, taken
together with the present structures of Erbin PDZ, demonstrate that the P2 site is a hitherto unrecognized important structural
element with possible regulatory function, at least for a subset of PDZ domains. Moreover, the emerging complexity of PDZ selectivity mechanisms points to the need for new PDZ classification schemes that
will take into consideration the
2-
3 loop length, the specificity of the P2-ligand interaction, and the structural
determinants underlying the dual ligand specificity of these versatile
protein modules.
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ACKNOWLEDGEMENTS |
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We thank the staff of the Macromolecular Diffraction Facility at the Cornell High Energy Synchrotron Source for assistance during data collection, and Drs. Verna Frasca and Lung-Nan Lin at MicroCal LLC for the calorimetric analysis.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health, the Massachusetts Department of Public Health, and the United States Department of Defense (to J. A. A. L.).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 structure factors (codes 1MFG and 1MFL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Molecular Medicine Laboratory,
Harvard Institutes of Medicine, Rm. 354, 4 Blackfan Circle, Boston, MA
02115. E-mail: jladias@caregroup.harvard.edu.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.C200571200
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ABBREVIATIONS |
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The abbreviations used are: PDZ, PSD-95/DLG/ZO-1; MAD, multiwavelength anomalous dispersion; SeMet, selenomethionine.
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