Novel Mode of Ligand Recognition by the Erbin PDZ Domain*

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 α-helix. The isopropyl group of valine at position −2 of the ErbB2 peptide interacts with the Erbin Val1351and displaces the peptide backbone away from the α-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 β2-β3 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.

PDZ 1 (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)(2)(3). The PDZ fold comprises a six-stranded antiparallel ␤-barrel capped by two ␣-helices (1)(2)(3)(4)(5)(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)(2)(3). The residues at positions 0 and Ϫ2 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 Ϫ5 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 Ϫ2 (1)(2)(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 ␦-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 Ϫ7 (corresponding to Tyr 1248 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 Ϫ7 has not been observed in previous structural studies (1-7). Importantly, phosphorylation of Tyr 1248 following ErbB2 activation is a critical event for the mitogenic signaling and oncogenicity of this receptor (14 -16). Moreover, Tyr 1248 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 Ϫ7 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 Ϫ7 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
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 fur-* 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The ther 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 SeMetsubstituted 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 P2 1 with unit cell dimensions a ϭ 26.6 Å, b ϭ 57.4 Å, c ϭ 30.4 Å, ␤ ϭ 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 P2 1 with a ϭ 26.5 Å, b ϭ 57.0 Å, c ϭ 30.9 Å, ␤ ϭ 99.2°. Data were processed using DENZO and SCALEPACK (18) (Table I).
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 REF-MAC (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 Glu 1280 , Ser 1294 , Ser 1325 , His 1347 , Gln 1349 , and Ile 1365 are modeled in two conformations. The main conformation of His 1347 (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
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 ␣-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.
The ErbB2 peptide inserts into the Erbin PDZ ligand-binding groove antiparallel to the ␤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 P 1 ), 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 Ϫ2 makes hydrophobic contacts with Val 1351 , 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 Ϫ3 with Thr 1316 (Fig. 1D), whereas Leu Ϫ4, Gly Ϫ5, and Leu Ϫ6 do not bind to PDZ. Interestingly, the imidazole ring of the conserved His 1347 , which is the hallmark of class I PDZ domains and plays a critical role in the selection of the residue Ϫ2, points away from Val Ϫ2, where it hydrogen bonds with the carbonyl oxygen of Gly 1299 (Fig. 1B).
The ␤2-␤3 Loop of Erbin PDZ Interacts with Tyr Ϫ7 of the ErbB2 Ligand-Strikingly, the phenolic ring of Tyr Ϫ7 folds back in a direction parallel to the peptidic backbone and enters a pocket, designated P 2 , which is formed by Ser 1296 in the ␤2 strand and Gly 1303 , Asn 1304 , and Pro 1305 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 Ϫ7. 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 Ϫ7 is stacked. The phenolic ring of Tyr Ϫ7 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 Ϫ7 hydrogen bonds through two ordered water molecules with Asp Ϫ3 (Fig. 1D).
Phosphorylation of Tyr Ϫ7 Abolishes Binding to the P 2 Pock- where I is the observed integrated intensity, ͗I͘ is the average integrated intensity obtained from multiple measurements, and the summation is over all observed reflections. d R cryst ϭ ⌺ʈF obs ͉ Ϫ k͉F calc ʈ/⌺͉F obs ͉, where F obs and F calc are the observed and calculated structure factors, respectively. e R free is calculated as R cryst using 5% of the reflection data chosen randomly and omitted from the refinement calculations.
et-Because phosphorylation of Tyr 1248 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 Ϫ5 to Ϫ8 and the P 2 pocket is empty ( Fig. 2A). In contrast, Val 0, Pro Ϫ1, Val Ϫ2, Asp Ϫ3, and Leu Ϫ4 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 K d of ϳ50 M, whereas the phosphotyrosine-containing peptide binds to PDZ with a K d of ϳ128 M. The ϳ2.5-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 Ϫ7 inside the P 2 pocket.
Superposition of the Erbin PDZ structures with the PSD-95 PDZ3 (4) reveals that Val 0, Pro Ϫ1, Val Ϫ2, and Asp Ϫ3 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 Ϫ2 interaction with Val 1351 rather than the Tyr Ϫ7 binding to P 2 . 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 P 2 site is preformed and does not undergo major conformational changes upon Tyr Ϫ7 binding. By contrast, the ␤2-␤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 P 2 to discriminate between the phosphorylation states of Tyr Ϫ7 indicates that it may play a regulatory role in ErbB2 signaling and suggests an attractive  (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 2F obs Ϫ F calc electron density map at the P 2 pocket calculated at 1.25 Å and contoured at 2.5 . model for this regulation. Conceivably, during the inactive state of ErbB2, Tyr Ϫ7 is buried inside P 2 and is inaccessible for phosphorylation and interaction with other proteins. Activation of ErbB2 triggers the release of Tyr Ϫ7 from P 2 , 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 Ϫ7. 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 Ϫ7 restores its original position inside P 2 . Importantly, in contrast to the regulatory site P 2 that oscillates between bound and unbound states, P 1 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 Cterminal region, because these modules have non-overlapping recognition motifs.
Do other PDZ domains have a P 2 pocket? In contrast to the short ␤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)(26)(27)(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 P 2 in PDZ-ligand interactions. These observations, taken together with the present structures of Erbin PDZ, demonstrate that the P 2 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 P 2ligand interaction, and the structural determinants underlying the dual ligand specificity of these versatile protein modules.