ACCELERATED PUBLICATION
Novel Mode of Ligand Recognition by the Erbin PDZ Domain*

Gabriel Birrane, Judy Chung, and John A. A. LadiasDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha -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 alpha -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 beta 2-beta 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
REFERENCES

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 beta -barrel capped by two alpha -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-Phi (X denoting any amino acid and Phi  representing a hydrophobic residue), whereas class II domains recognize the motif X-Phi -X-Phi (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).

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 delta -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).

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 -7 with the extended beta 2-beta 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 beta 2-beta 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

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, beta  = 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 CuKalpha radiation. The crystals belong to space group P21 with a = 26.5 Å, b = 57.0 Å, c = 30.9 Å, beta  = 99.2°. Data were processed using DENZO and SCALEPACK (18) (Table I).

                              
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Table I
Statistics of structure determination and refinement

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).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha -helix that is present between the beta 3 and beta 4 strands in PDZs with known structure (1-7), due to the shorter length of the Erbin beta 3-beta 4 loop (Fig. 1, A and B). The significance of this deviation from the canonical PDZ fold is unclear because this alpha -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 alpha -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 sigma .

The ErbB2 peptide inserts into the Erbin PDZ ligand-binding groove antiparallel to the beta 2 strand, extending and twisting the beta -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 alpha -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).

The beta 2-beta 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 beta 2 strand and Gly1303, Asn1304, and Pro1305 in the beta 2-beta 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 beta 2-beta 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).

Phosphorylation of Tyr -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 sigma  is superimposed on the ErbB2 peptide. B, superposition of the Calpha 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.

Superposition of the Erbin PDZ structures with the PSD-95 PDZ3 (4) reveals that Val 0, Pro -1, Val -2, and Asp -3 are superposed extremely well in both Erbin complexes, whereas the ErbB2 backbone is displaced away from the alpha -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 beta 2-beta 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 beta 2-beta 3 loops of the Erbin PDZ and PSD-95 PDZ3 occupy completely different positions and are not superimposable.

Structural and Functional Implications-- The property of the newly discovered pocket P2 to discriminate between the phosphorylation states of Tyr -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.

Do other PDZ domains have a P2 pocket? In contrast to the short beta 2-beta 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 beta 2-beta 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 beta 2-beta 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 beta 2-beta 3 loop length, the specificity of the P2-ligand interaction, and the structural determinants underlying the dual ligand specificity of these versatile protein modules.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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/).

Dagger 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

    ABBREVIATIONS

The abbreviations used are: PDZ, PSD-95/DLG/ZO-1; MAD, multiwavelength anomalous dispersion; SeMet, selenomethionine.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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