PDZ protein interaction domains are typically selective for C-terminal ligands, but non-C-terminal, 'internal' ligands have also been identified. The PDZ domain from the cell polarity protein Par-6 binds C-terminal ligands and an internal sequence from the protein Pals1/Stardust. The structure of the Pals1−Par-6 PDZ complex reveals that the PDZ ligand-binding site is deformed to allow for internal binding. Whereas binding of the Rho GTPase Cdc42 to a CRIB domain adjacent to the Par-6 PDZ regulates binding of C-terminal ligands, the conformational change that occurs upon binding of Pals1 renders its binding independent of Cdc42. These results suggest a mechanism by which the requirement for a C terminus can be readily bypassed by PDZ ligands and reveal a complex set of cooperative and competitive interactions in Par-6 that are likely to be important for cell polarity regulation.
Protein interaction domains form the backbone of cellular information flow1. The PDZ protein interaction domain participates in a wide variety of signaling pathways and is one of the most common in metazoan genomes2. Because they often occur in multiple instances in the same polypeptide, PDZ domains are thought to have an organizational role in signal transduction pathways3. Given the large number of PDZ domains, several modes of ligand recognition exist whose mechanisms are still being elucidated.
PDZ domains bind to short sequences of five to seven residues in their target proteins4,
5. Although these recognition sequences have a low information content, specificity is typically enhanced by the requirement that the sequence occurs at the C terminus. C-terminal recognition falls into several different classes, depending on the identity of critical binding residues3. For example, class I PDZ ligands have a consensus sequence of Ser-X-Val-COOH, where X indicates any residue.
The requirement for a C terminus results from a steric rather than an electrostatic mechanism. The peptide-binding pocket is constructed such that residues that extend past the C terminus clash with a conserved PDZ segment known as the carboxylate-binding loop3,
4. Although C-terminal ligands have a negatively charged carboxylate, studies of salt effects on the binding reaction suggest that electrostatic contributions are negligible6. Because of the additional specificity provided by the C terminus, enforcement of C-terminal binding is an important component of PDZ-ligand recognition.
Although recognition of C-terminal motifs seems to be the dominant mode of PDZ-ligand interaction, non-C-terminal (also known as internal), PDZ ligands also exist. Compared with our understanding of C-terminal PDZ ligands, however, the mechanism of internal ligand recognition is much less clear. The best-characterized internal PDZ interactions involve ligands that adopt a specific conformation that adheres to the steric requirements of the PDZ-binding pocket. For example, in the hetero-oligomerization of the nNOS and syntrophin PDZ domains, an extension of the nNOS PDZ domain, termed the -finger, forms a sharp -turn where the C terminus would occur in a C-terminal PDZ ligand7. Likewise, an internal PDZ ligand identified in a phage display library contains a disulfide that presumably allows the chain to avoid the steric block as reduction of the disulfide abrogates binding8. The specialized sequences of these internal ligands suggest that bypassing the requirement for a C terminus requires the ligand to adopt a specific, preformed conformation. For example, the nNOS PDZ domain is required for high-affinity binding to the syntrophin PDZ as the -finger is stabilized through interactions with the domain9.
Several questions about recognition of internal sequences by PDZ domains remain. First, can PDZ domains bind to internal sequences that don't have specialized structures such as the nNOS -finger? PDZs have been shown to bind internal sequences that don't themselves contain PDZs10, indicating that PDZ oligomerization is not the only mechanism for internal binding. Are these alternative internal ligands as dependent on conformation as the -finger of nNOS-syntrophin? Answering these questions will allow us to better understand the diversity of ligand interactions in this important protein interaction domain family.
The cell polarity protein Par-6 is an excellent model system for studying internal PDZ recognition. Par-6 contains a single PDZ domain that has been shown to bind to both internal and C-terminal ligands. Par-6 binds to the C terminus of the transmembrane receptor Crumbs (Crb)11 and a similar C-terminal sequence identified in a peptide library screen12. Par-6 binding to C-terminal ligands is regulated by binding of the Rho GTPase Cdc42 to a CRIB domain adjacent to the Par-6 PDZ. An allosteric transition in the CRIB-PDZ of Par-6 induced by Cdc42 binding leads to conversion from a low-affinity to a high-affinity PDZ conformation12. In addition, Par-6 has recently been shown to bind to partner of Lin-7 (Pals1) and its Drosophila melanogaster homolog Stardust (Sdt) through an internal interaction that is important for formation of tight junctions in epithelial cells10.
To further understand PDZ internal recognition, we examined the interaction of the Par-6 PDZ with Pals1. Binding studies reveal that a markedly small sequence from Pals1 is sufficient for binding to the Par-6 PDZ domain; this is inconsistent with the internal binding mode in nNOS-syntrophin. In addition, Cdc42 binding to Par-6 has no effect on its affinity for Pals1, in contrast to C-terminal ligands. Comparison of the Par-6−Pals1 internal complex with a C-terminal complex reveals that binding of Pals1 deforms the PDZ-binding pocket, thereby bypassing the enforcement of C-terminal binding, and presumably decoupling binding to Cdc42. These results reveal a conformational plasticity of PDZ domains that can be exploited for both recognition and regulation.
Results A small internal Par-6 PDZ ligand in Pals1/Sdt Par-6 has been shown to bind Pals1 through an interaction with the Par-6 PDZ domain (Fig. 1a)10. To examine the mechanism of this interaction, we identified the minimal components of Pals1, and its D. melanogaster homolog Stardust (Sdt), that are necessary for binding. Pals1 and Sdt have domain structures typical of other members of the MAGUK (membrane-associated guanylate kinase) protein family with PDZ and SH3-GK protein modules13 (Fig. 1). The region of Pals1 that binds to Par-6 is in a region N-terminal to these domains10. Further deletion analysis of the Pals1 N terminus reveals that a small eight-residue motif is sufficient for binding to the Par-6 PDZ domain (Fig. 1b). Two similar motifs are present in Sdt, both of which also bind to the Par-6 PDZ (Fig. 1b,c). The nonredundant sequence database contains several sequences related to the eight-residue Pals1 sequence from several metazoan organisms. Alignment of these sequences reveals a core motif in the Pals1/Sdt Par-6 ligand sequence that is highly conserved (Fig. 1d). This core motif is necessary and sufficient for binding to Par-6 (Fig. 1e).
Figure 1. Identification of Par-6 PDZ-binding motifs in Pals1 and Stardust.
(a) Schematic of Par-6 domain structure and domain ligands (PB1, Phox and Bem1; CRIB, Cdc42/Rac interactive binding; PDZ, PSD-95, discs large, ZO-1). (b) Deletion analysis of Pals1 and Sdt reveals a short, internal Par-6 PDZ ligand. GST fusions of various murine Pals1 or D. melanogaster Stardust fragments were tested for their ability to bind Par-6 using glutathione agarose affinity chromatography (Palspep, Pals1 residues 29−40; Sdt2pep, Sdt residues 375−388). (c) Domain structure of Pals1 and Sdt showing the relationship of the Par-6 PDZ internal ligands to the MAGUK protein domains (L27, Lin-2, Lin-7; SH3, Src homology 3; GK, guanylate kinase homology). (d) Alignment of Pals1/Sdt Par-6 ligand sequences showing conserved core sequence. (e) Deletion analysis of Pals1/Sdt Par-6 ligand sequences. The conserved core sequence is required for binding to the Par-6 PDZ domain. (f) The Rho GTPase Cdc42 does not regulate Pals1 binding to the Par-6 PDZ domain. A rhodamine-labeled peptide with the Pals1 sequence was used to measure binding to the Par-6 CRIB-PDZ fragment. The higher anisotropy of the Cdc42 complex at saturation occurs because of its higher molecular mass. The binding curves are for Kd values of 6 and 8 M for Cdc42-bound and free Par-6, respectively.
The small size of the Pals1/Sdt internal ligand indicates that its binding mode differs from that of the nNOS-syntrophin internal interaction. Because the nNOS -finger is structurally coupled to the adjacent nNOS PDZ domain, >100 residues from nNOS are required for binding to the syntrophin PDZ9. The eight residues sufficient for the Pals1−Par-6 interaction are more similar to canonical C-terminal interactions that require approximately six residues5. However, the region of Pals1 that binds to Par-6 does not occur at its C terminus, and therefore must use a different mechanism of recognition.
We also find that binding of the Pals1/Sdt internal ligand is independent of the interaction of Par-6 with the Rho GTPase Cdc42. Cdc42 binds to a CRIB (Cdc42/Rac interactive binding) motif adjacent to the Par-6 PDZ domain and increases the affinity of the PDZ domain for C-terminal ligands 13-fold (ref. 12). Previous qualitative data with a large fragment of Pals1 indicated that Cdc42 does not affect the Par-6−Pals1 interaction12. To test the possible coupling of Cdc42 and Pals1 binding in a quantitative fashion, we synthesized the Pals1-binding sequence with an NH2-terminal rhodamine and measured binding using fluorescence anisotropy. Cdc42 binding to Par-6 has no effect on the affinity of Par-6 for Pals1 (Fig. 1e).
The small size of the Pals1 ligand, which is inconsistent with the formation of a specialized structure, and the lack of Cdc42 regulation of Pals1 binding lead to several questions about the mechanism of Par-6 recognition of this ligand. In particular, how does the short Pals1 sequence satisfy the steric restraints of the PDZ-binding pocket? Also, given that Pals1 and C-terminal ligands compete for binding to Par-6 (ref. 12), how is it that the Par-6−Pals1 interaction is independent of Cdc42 binding?
Crystal structure of the Par-6 PDZ−Sdt/Pals1 complex To address the mechanism of internal recognition by Par-6−Pals1/Sdt, we determined the structure of the Par-6 PDZ in complex with the minimal core motif using X-ray crystallography. The D. melanogaster Par-6 PDZ domain (residues 156−255) was cocrystallized with the Sdt/Pals1-derived peptide Ac-YPKHREMAVDCP-CONH2 (representing amino acids 29−40 from Pals1) and formation of crystals was dependent on the presence of peptide. Crystals grew in the space group R3 with one complex per asymmetric unit and diffracted to 2.5 Å (diffraction was somewhat anisotropic; see Methods). Phases were determined by molecular replacement using the PDZ domain from the Par-6 PDZ−C-terminal peptide complex (PDB entry 1RZX) as a search model. The Pals1 peptide was clearly visible in the initial electron density maps. The refined model contains residues 156−255 from Par-6, residues 30−40 of Pals1, and five waters, and has an R-factor of 21% and an Rfree of 25%. Simulated annealing omit maps for both the bound peptide and carboxylate binding loop are shown in Supplementary Figure 1 online.
In the structure of the Par-6−Pals1 complex, the Pals1 ligand occupies the same binding site as C-terminal ligands. C-terminal ligands bind in a groove that is formed between strand 2 and helix 2 of the PDZ domain3,
4. The HREMAV portion of Pals1 lies in this same groove, corresponding to the equivalent residues of the C-terminal ligand (VKESLV-COOH). This is similar to the nNOS-syntrophin internal PDZ interaction in which a sequence in the nNOS -finger mimics C-terminal ligands (this sequence is known as the pseudopeptide) and occupies the same binding site as C-terminal ligands7. The most critical residues for Par-6 PDZ C-terminal ligands are the P(0) valine (the C-terminal residue), the P(-1) leucine, and the P(-3) aspartic acid12. Although the P(0) valine is conserved in Pals1, the P(-1) leucine is replaced by an alanine. Previous alanine scanning studies have shown that an alanine at this position abrogates binding in the context of the C-terminal ligand12, indicating that although the general features of pseudopeptide recognition in the Sdt/Pals1 internal ligand are similar, there are notable differences.
Internal recognition through PDZ domain plasticity Binding of Pals1 induces a conformational change in the carboxylate-binding loop of the Par-6 PDZ domain. In comparison to the structure of the Par-6 PDZ in complex with a C-terminal ligand, the overall fold of the PDZ domain is the same (Fig. 2a). However, two loops that connect the 1 and 2 strands, and the 2 and 3 strands, contain substantial deviations between the two structures, with distances between backbone atoms of >3 Å, and up to 7 Å in the carboxylate-binding loop. The altered conformation of the carboxylate-binding loop occurs between residues 163 and 171 (note that these residues make no significant crystal contacts). Residue 171 is the first residue of the 'GLGF' loop, a set of conserved residues in PDZ domains that make up the base of the carboxylate-binding loop. However, in Par-6 this residue is a proline rather than a glycine. Mutation of this proline in Par-6 to the canonical glycine leads to disruption of regulated C-terminal binding but does not alter internal binding12. The peptide backbone of this loop forms contacts with the carboxylate from C-terminal ligands and therefore plays a central role in enforcing C-terminal binding3,
4. The other loop that differs between the two complexes is a large loop connecting strands 2 and 3. As this loop makes extensive interdomain contacts due to crystal packing and does not significantly contact the PDZ ligand, it is likely that the difference in this loop is not important for internal recognition.
Figure 2. Comparison of Par-6 PDZ internal and C-terminal binding.
(a) Comparison of Par-6 PDZ domains from the Pals1 complex and the C-terminal peptide complex (PDB entry 1RZX). The Pals1 complex is yellow and the C-terminal complex is cyan with the secondary structure elements labeled. The ligands have been removed for clarity. The distance between equivalent C atoms in the C-terminal peptide and Pals1 bound Par-6 PDZ domains is plotted below the structures. (b) Par-6 PDZ in complex with a C-terminal peptide. Binding of C-terminal ligands is enforced by the carboxylate-binding loop (colored cyan on the PDZ surface) through a steric mechanism in which residues that would extend past the P(0) residue would clash with the loop. (c) Par-6 PDZ in complex with an internal peptide from Pals1. When bound to the Pals1 peptide, the carboxylate-binding loop (colored yellow on the PDZ surface) is deformed, allowing it to extend past the P(0) residue.
The conformational change in the Par-6 PDZ carboxylate-binding loop allows the Pals1 ligand to bypass the requirement for a C terminus. The carboxylate-binding loop caps the PDZ-binding pocket, preventing binding of ligands that continue past the P(0) residue (Fig. 2b). This loop has a similar conformation in free PDZ domain structures4. The conformational change that occurs in the carboxylate-binding loop substantially alters this region of the binding pocket, however (Figs. 2b,c and 3). A lysine from the carboxylate-binding loop (Lys165) is positioned closer to the aspartic acid side chain (Fig. 3) and the interaction between these two residues is critical for Pals1 binding (see below). Additionally, hydrogen bonding interactions between the carboxylate and the PDZ backbone (Fig. 3a) are now replaced with backbone-backbone interactions in the Par-6−Pals1 complex (Fig. 3b).
Figure 3. Critical interactions in Pals1 internal PDZ binding.
(a) Par-6 PDZ−C-terminal ligand interactions. The peptide-binding pocket from the C-terminal peptide−Par-6 PDZ complex (PDB entry 1RZX) is shown. Peptide residues are labeled by amino acid and PDZ domain residues are labeled by amino acid and sequence number. The distance between the C terminus and Lys165 is shown (solid line) along with interactions between the carboxylate and the PDZ backbone (dashed lines). (b) Par-6 PDZ−Pals1 internal ligand interactions. The interactions between the PDZ domain and peptide are shown as in a.
The rearrangement of the Par-6 PDZ carboxylate-binding loop to allow for Pals1 binding differs from the situation in nNOS-syntrophin. In the nNOS-syntrophin interaction, the structure of the syntrophin PDZ domain is not altered when nNOS binds7. Instead, the ligand adapts to the requirements enforced by the syntrophin PDZ carboxylate-binding loop by forming a sharp turn that avoids the steric constraints imposed by the loop.
The ability of Pals1 to bind internally does not result solely from the residues that follow the P(0) valine. A peptide containing the sequence of the C-terminal ligand, followed by the residues from Pals1 that follow the P(0) valine (Asp-Cys-Pro) does not bind to Par-6 (data not shown), indicating that there is an interplay between residues before and after the P(0) valine. This interplay arises because of differences in the way the P(-1)-binding pocket is used in the two types of ligands (see below).
Determinants of internal and Cdc42-regulated binding A critical question in understanding Par-6 PDZ internal versus C-terminal recognition is why the internal ligand can deform the carboxylate-binding loop whereas the C-terminal ligand cannot. In other words, why is a C terminus required in one class of ligands, but not for internal ligands? To answer this question, we generated a series of substitutions in the Pals1 sequence and tested their ability to bind to the Par-6 PDZ domain.
Alanine scanning of the Pals1 internal ligand reveals a different pattern of required residues than Par-6 PDZ C-terminal ligands. We had previously identified the C-terminal ligand VKESLV-COOH as a Par-6 PDZ ligand12, and the C terminus of the transmembrane protein Crumbs (Crb) with the sequence EERLI-COOH has also been shown to bind11. From these data, the pattern of required residues for Par-6 PDZ C-terminal ligands is XEXLV/I-COOH (where X indicates that the residue can be replaced by alanine without substantially altering binding). We generated alanine substitutions of the Pals1/Sdt ligand and tested their ability to bind the Par-6 PDZ domain in a qualitative pull-down assay (Fig. 4a). These results indicate that the pattern of required residues in the Pals1 ligand is XEXAVDX. Whereas C-terminal ligands require a large hydrophobic residue at the P(-1) position for binding, the Pals1 internal ligand has an alanine at this position. This is because the residues following the P(0) valine (predominantly the P(+2) cysteine) occupy the binding site that P(-1) leucine occupies in C-terminal ligands.
Figure 4. Analysis of Par-6 PDZ ligand-binding determinants.
(a) Alanine scanning of Pals1 ligand. GST fusions of peptide sequences with an alanine substituted at each position along the Pals1 sequence were tested for their ability to bind the Par-6 PDZ domain. (b) Evaluation of the importance of the P(+1) residue in Pals1. In the structure of the Par-6−Pals1 complex, the P(+1) aspartic acid forms a salt bridge with a conserved lysine in the PDZ domain. GST fusions of mutants in this position were tested for their ability to bind the Par-6 PDZ domain. Mutation to an asparagine abrogated binding, indicating that the salt bridge is critical for binding. Bold residues indicate site of mutation. (c) Identifying the determinants of Cdc42 regulation. As has been previously shown, Cdc42 binding to the CRIB-PDZ fragment of Par-6 alters its affinity for C-terminal ligands. GST fusions of several Pals1 truncations were made to identify the determinants of Cdc42 regulation. When the P(+1) residue was removed (HREMAV sequence), Cdc42 binding caused an increase in peptide binding. The presence of this residue (HREMAVD sequence) caused Cdc42 to no longer affect peptide binding, presumably because the carboxylate-binding loop was deformed.
In the structure of the Par-6 PDZ−Pals1 complex, the P(+1) aspartic acid seems to form a salt bridge with a conserved lysine in the carboxylate-binding loop. This lysine forms interactions with the C-terminal carboxylate through a series of water molecules in C-terminal ligands4. As the alanine scanning showed that this aspartic acid is crucial for binding, we made further mutations in this residue to test the requirement for a carboxylate at this position. Removal of the carboxylic acid by mutation of the aspartic acid to asparagine completely disrupted binding (Fig. 4b). Considering also the alanine scanning results showing that residues following this residue are not critical, we conclude that the P(+1) aspartic acid is a key determinant for internal binding. The ion pairing interaction with a conserved PDZ lysine side chain may be important for stabilizing the deformed conformation of the carboxylate-binding loop.
A key difference between binding of C-terminal and internal ligands is that C-terminal ligands are regulated by Cdc42 whereas internal ligands are not. To investigate the sequence determinants of the two ligands that lead to this difference, we made a series of deletions in the Pals1 ligand and tested their ability to bind Par-6 in a Cdc42-regulated manner. A ligand that is truncated to the P(0) valine bound with higher affinity in the presence of Cdc42 (Fig. 4c). However, a ligand that extends one residue beyond this position to the P(+1) aspartic acid bound to the Par-6 PDZ domain in a Cdc42-independent manner. This suggests that Cdc42-regulated binding is dependent on the normal conformation of the carboxylate-binding loop and that the deformation of this loop by the Pals1 ligand decouples the two binding sites.
Discussion PDZ domains are typically selective for C-terminal ligands, a restraint imposed by the conformational properties of their binding pockets (Fig. 5). Internal interactions that bypass the requirement for a C terminus have been thought to be restricted to ligands that form specialized conformations that adapt to the properties of the PDZ-binding pocket3,
7 (Fig. 5). We have demonstrated that Par-6 interacts with Pals1/Sdt through an internal PDZ-peptide interaction that deforms the PDZ carboxylate-binding loop (Fig. 5). This mechanism of internal binding indicates that internal PDZ ligands do not require the specific conformational constraints of the nNOS -finger, suggesting that internal ligands may be more common than previously thought3. Comparison of PDZ sequences to the Par-6 PDZ domain does not reveal any obvious differences from other PDZ domains that would suggest that it is unique in its ability to bind Pals1-type internal ligands. Further work will be required to establish whether the mode of internal binding described here is a property specific to the Par-6 PDZ domain, or if a large subset of PDZ domains can use this mechanism.
Figure 5. Modes of PDZ C-terminal and internal recognition.
In PDZ C-terminal ligand recognition, the carboxylate-binding loop enforces C-terminal binding by preventing extension past the P(0) residue. In the -finger internal PDZ recognition mode of recognition, used by nNOS-syntrophin7 and presumably disulfide-containing ligands8, a sharp turn in the ligand allows it to bypass the steric requirement imposed by the carboxylate-binding loop. The Pals1−Par-6 PDZ interaction represents a new type of internal interaction in which the carboxylate-binding loop is deformed to allow for extension past the P(0) residue. An interaction with the P(+1) residue is critical for this mode of recognition.
In the first structure of a PDZ domain with a C-terminal ligand4, the authors noted some plasticity in the carboxylate-binding loop upon ligand binding. The mode of internal sequence recognition described here may be an extension of this mechanism. However, it is clear that, at least in the case of the Par-6−Pals1 interaction, specific interactions beyond the P(0) residue are required to take advantage of carboxylate-binding loop plasticity. It is likely that the deformation of the carboxylate-binding loop is energetically costly, and additional interactions are required to compensate for this.
The plasticity of the Par-6 PDZ domain may be important for regulation of the large number of Par-6 binding partners, including Cdc42, Par-3, Pals1/Sdt, Crumbs and Lgl. We have shown that these ligands represent a complex set of cooperative and competitive binding partners. For example, binding of the C-terminal ligand Crumbs and the internal Pals1/Sdt are competitive with each other12, whereas the Pals1/Sdt PDZ domain itself has been shown to bind to Crumbs14. All three of these proteins are present in epithelial cells10,
15, so understanding the partitioning of possible complexes, and the role of these complexes in establishing and maintaining cell polarity, will require further in vitro and in vivo analysis.
We have shown previously that C-terminal ligand binding to the Par-6 PDZ is regulated by Cdc42 (ref. 12). Here we show that deformation of the Par-6 PDZ by Pals1 not only allows for internal recognition, but also renders Pals1 binding independent of Cdc42. These results suggest that coupling between Cdc42 and C-terminal ligand binding occurs predominantly through the carboxylate-binding loop, although other structural changes occur upon Cdc42 binding12. As Cdc42 binds to a region directly adjacent to the carboxylate-binding loop, the loop is well positioned to transduce Cdc42 binding to the peptide-binding pocket.
In summary, we have identified an internal ligand for the Par-6 PDZ domain and shown that it uses a distinct mode of recognition from previously studied internal interactions. Rather than adapting to the specific conformation of the PDZ-binding pocket, the Par-6 PDZ domain is deformed to bypass the requirement for chain termination. We have also shown that the two different PDZ conformations have different regulatory properties, with only C-terminal ligands being regulated by Cdc42. These results provide a framework for understanding diverse PDZ-ligand interactions.
Methods Protein expression and purification. We purified poly-His fusions using Ni-NTA resin followed by incubation with TEV protease to remove the His-tag. Tag cleavage was followed by further purification using ion exchange chromatography to achieve a final purity of >99% as measured by SDS-PAGE and/or MALDI-TOF mass spectrometry.
In vitro binding assays. For GST pull-down assays, we adsorbed the GST fusion onto glutathione agarose beads and washed with binding buffer (10 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.05% (v/v) Triton, pH 7.5). We then added ligands at the indicated concentration and incubated at room temperature for 15 min. We removed unbound protein by washing three times with a large excess of binding buffer. After elution of GST fusions and bound proteins by addition of SDS loading buffer, we visualized the results of the binding assays by SDS-PAGE.
Rhodamine-labeled Pals1 peptide for fluorescence anisotropy was prepared using FMOC solid phase synthesis. We coupled the rhodamine (rhodamine B, Sigma) to the peptide NH2-terminal amine as the last step of the synthesis before the peptide was cleaved from the resin. We purified the peptide by reverse-phase HPLC and verified its identity by mass spectrometry. Fluorescence anisotropy binding assays were carried out on an ISS PC1 fluorimeter and the data were fit to an equation describing a bimolecular binding reaction with peptide concentration below the Kd of the interaction.
Crystallization and structure determination. The Par-6 PDZ domain (from D. melanogaster, residues 156−255) was crystallized by hanging-drop vapor diffusion at 15 °C using 26% (w/v) PEG6000, 100 mM HEPES, pH 7.1, as a mother liquor. We mixed the Par-6 PDZ domain, concentrated to 10 mg ml-1, with a three-fold molar excess of peptide representing Pals1(29−40) (NH2-terminal acetylated and with a COOH-terminal amide). Crystals grew in space group R3 with cell constants of a = b = 63.1 Å, c = 99.3 Å and one PDZ−Pals1 peptide complex per asymmetric unit.
Crystals were cooled in liquid nitrogen using mother liquor plus 10% (v/v) glycerol as a cryoprotectant. Diffraction data was collected at beamline 8.2.2 at the Advanced Light Source using an ADSC Q315 CCD detector. Crystals diffracted somewhat anisotropically, ranging between 2.5 and 2.7 Å. Data reduction was carried out using the HKL2000 package16. Crystallographic data statistics are shown in Table 1.
Table 1. Data collection and refinement statistics
The coordinates of the Par-6 PDZ domain from the Par-6 PDZ−C-terminal peptide complex (PDB entry 1RZX) were used as a search model for molecular replacement using the AmoRe17. A single solution was found that provided initial phases that were further improved with rigid body optimization using CNS18. Initial electron density maps showed clear density in the peptide-binding pocket. The initial model was refined with alternate cycles of positional and restrained B-factor refinement followed by manual rebuilding using composite simulated annealing omit maps in O19. To further improve the model, additional TLS refinement using REFMAC20 was done using the same set of test reflections as in CNS18. Two TLS groups were defined that included the PDZ domain and the Pals1 ligand, respectively. TLS and restrained refinement resulted in a final model with an R of 0.216 and Rfree of 0.254 and excellent geometry (Table 1). Additional information about the model, including simulated annealing electron density maps, B-factor distributions and real space correlation coefficients can be found in Supplementary Figures 1,2,3,4, online.
Received 18 May 2004; Accepted 13 September 2004; Published online: 10 October 2004.
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Acknowledgments We thank A. Berglund, T. Stevens, B. Volkman, and members of the Prehoda Lab for helpful comments and suggestions. We thank the support staff at beamline 8.2.1 at the Advanced Light Source for technical assistance. This work was supported by grants from the American Heart Association, National Institutes of Health (GM068032), and a Damon Runyon Scholar Award to K.E.P.
Competing interests statement:
The authors declare that they have no competing financial interests.
-finger, forms a sharp 
M for Cdc42-bound and free Par-6, respectively.
13-fold (ref. 
atoms in the C-terminal peptide and Pals1 bound Par-6 PDZ domains is plotted below the structures. (b) Par-6 PDZ in complex with a C-terminal peptide. Binding of C-terminal ligands is enforced by the carboxylate-binding loop (colored cyan on the PDZ surface) through a steric mechanism in which residues that would extend past the P(0) residue would clash with the loop. (c) Par-6 PDZ in complex with an internal peptide from Pals1. When bound to the Pals1 peptide, the carboxylate-binding loop (colored yellow on the PDZ surface) is deformed, allowing it to extend past the P(0) residue.
