PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide

The Escherichia coli protease HtrA has two PDZ domains, and sequence alignments predict that the E. coli protease Tsp has a single PDZ domain. PDZ domains are composed of short sequences (80-100 amino acids) that have been implicated in a range of protein:protein interactions. The PDZ-like domain of Tsp may be involved in binding to the extreme COOH-terminal sequence of its substrate, whereas the HtrA PDZ domains are involved in subunit assembly and are predicted to be responsible for substrate binding and subsequent translocation into the active site. E. coli has a system of protein quality control surveillance mediated by the ssrA-encoded peptide tagging system. This system tags misfolded proteins or protein fragments with an 11-amino acid peptide that is recognized by a battery of cytoplasmic and periplasmic proteases as a degradation signal. Here we show that both HtrA and Tsp are able to recognize the ssrA-encoded peptide tag with apparent K(D) values of approximately 5 and 390 nm, respectively, and that their PDZ-like domains mediate this recognition. Fusion of the ssrA-encoded peptide tag to the COOH terminus of a heterologous protein (glutathione S-transferase) renders it sensitive to digestion by Tsp but not HtrA. These observations support the prediction that the HtrA PDZ domains facilitate substrate binding and the differential proteolytic responses of HtrA and Tsp to SsrA-tagged glutathione S-transferase are interpreted in terms of the structure of HtrA.     

Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain

The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assays to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.     

Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.     

Surface plasmon resonance analysis of the binding of high‐risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI‐1

The E6 oncoproteins from high‐risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain‐containing proteins. Human MAGI‐1 is a multi‐PDZ domain protein implicated into protein complex assembly at cell–cell contacts. High‐risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI‐1 via a C‐terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione‐S‐transferase (GST). This approach was applied to measure the binding of MAGI‐1 PDZ1 to the C‐termini of viral or cellular proteins. Both high‐risk mucosal HPV E6 C‐terminal peptides and cellular partners of MAGI‐1 PDZ1 bind to MAGI‐1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI‐1 PDZ1 shows a preference for C‐termini with a valine at position 0 and a negative charge at position ?3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site‐directed mutagenesis of the HPV16 C‐terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ‐binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K₄₉₉ residue of MAGI‐1 as a novel determinant of binding specificity. Finally, we showed that MAGI‐1 PDZ1 also binds to the C‐termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI‐1. Copyright © 2010 John Wiley & Sons, Ltd.     

Peptide binding to the PDZ3 domain by conformational selection

The PDZ domains, a large family of peptide recognition proteins, bind to the C‐terminal segment of membrane ion channels and receptors thereby mediating their localization. The peptide binding process is not known in detail and seems to differ among different PDZ domains. For the third PDZ domain of the synaptic protein PSD‐95 (PDZ3), a lock‐and‐key mechanism was postulated on the basis of the almost perfect overlap of the crystal structures in the presence and absence of its peptide ligand. Here, peptide binding to PDZ3 is investigated by explicit solvent molecular dynamics (MD) simulations (for a total of 1.3 μs) and the cut‐based free energy profile method for determining free energy barriers and basins. The free energy landscape of apo PDZ3 indicates that there are multiple basins within the native state. These basins differ by the relative orientation of the α2 helix and β2 strand, the two secondary structure elements that make up the peptide binding site. Only the structure with the smallest aperture of the binding site is populated in the MD simulations of the complex whose analysis reveals that the peptide ligand binds to PDZ3 by selecting one of three conformations. Thus, the dynamical information obtained by the atomistic simulations increment the static, that is, partial, picture of the PDZ3 binding mechanism based on the X‐ray crystallography data. Importantly, the simulation results show for the first time that conformational selection is a possible mechanism of peptide binding by PDZ domains in general. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

The N-terminus of the WD5 repeat of human RACK1 binds to airway epithelial NHERF1

Regulation of the CFTR Cl channel function involves a protein complex of activated protein kinase Cepsilon (PKCepsilon) bound to RACK1, a receptor for activated C kinase, and RACK1 bound to the human Na(+)/H(+) exchanger regulatory factor (NHERF1) in human airway epithelial cells. Binding of NHERF1 to RACK1 is mediated via a NHERF1-PDZ1 domain. The goal of this study was to identify the binding motif for human NHERF1 on RACK1. We examined the site of binding of NHERF1 on RACK1 using peptides encoding the seven WD40 repeat units of human RACK1. One WD repeat peptide, WD5, directly binds NHERF1 and the PDZ1 domain with similar EC(50) values, blocks binding of recombinant RACK1 and NHERF1, and pulls down endogenous RACK1 from Calu-3 cell lysate in a dose-dependent manner. The remaining WD repeat peptides did not block RACK1-NHERF1 binding. An 11-amino acid peptide encoding a site on the PDZ1 domain blocks binding of the WD5 repeat peptide with the PDZ1 domain. An N-terminal 12-amino acid segment of the WD5 repeat peptide, which comprises the first of four antiparallel beta-strands, dose-dependently binds to the PDZ1 domain of NHERF1 and blocks binding of the PDZ1 domain to RACK1. These results suggest that the binding site might form a beta-turn with topology sufficient for binding of NHERF1. Our results also demonstrate binding of NHERF to RACK1 at the WD5 repeat, which is distinct from the PKCepsilon binding site on the WD6 repeat of RACK1.     

Consensus substrate sequence for protein-tyrosine phosphatase receptor type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.     

Deletion of the prc (tsp) gene provides evidence for additional tail-specific proteolytic activity in Escherichia coli K-12

The Escherichia coli protease Prc (Tsp) exhibits specificity in vitro for proteins with nonpolar carboxyl termini. To determine whether Prc is responsible for the selective degradation in vivo of proteins with nonpolar carboxyl termini, we constructed a prc (tsp) deletion strain. Deletion of the prc gene did not prevent the rapid intracellular degradation of a variant of the amino-terminal domain of λ repressor with a nonpolar carboxyl terminus, even though this protein is a substrate for Prc in vitro. Our results indicate that at least one additional carboxy-terminal-specific proteolytic system must exist in E. coli.     

Energetic determinants of internal motif recognition by PDZ domains

PDZ domains are protein-protein interaction modules that organize intracellular signaling complexes. Most PDZ domains recognize specific peptide motifs followed by a required COOH-terminus. However, several PDZ domains have been found which recognize specific internal peptide motifs. The best characterized example is the syntrophin PDZ domain which, in addition to binding peptide ligands with the consensus sequence -E-S/T-X-V-COOH, also binds the neuronal nitric oxide synthase (nNOS) PDZ domain in a manner that does not depend on its precise COOH-terminal sequence. In the structure of the syntrophin-nNOS PDZ heterodimer complex, the two PDZ domains interact in a head-to-tail fashion, with an internal sequence from the nNOS PDZ domain binding precisely at the peptide binding groove of the syntrophin PDZ domain. To understand the energetic basis of this alternative mode of PDZ recognition, we have undertaken an extensive mutagenic and biophysical analysis of the nNOS PDZ domain and its interaction with the syntrophin PDZ domain. Our data indicate that the presentation of the nNOS internal motif within the context of a rigid beta-hairpin conformation is absolutely essential to binding; amino acids crucial to the structural integrity of the hairpin are as important or more important than residues that make direct contacts. The results reveal the general rules of PDZ recognition of diverse ligand types.     

Structural Basis for Exquisite Specificity of Affinity Clamps, Synthetic Binding Proteins Generated through Directed Domain-interface Evolution

We have established a new protein-engineering strategy termed “directed domain-interface evolution” that generates a binding site by linking two protein domains and then optimizing the interface between them. Using this strategy, we have generated synthetic two-domain “affinity clamps” using PDZ and fibronectin type III (FN3) domains as the building blocks. While these affinity clamps all had significantly higher affinity toward a target peptide than the underlying PDZ domain, two distinct types of affinity clamps were found in terms of target specificity. One type conserved the specificity of the parent PDZ domain, and the other increased the specificity dramatically. Here, we characterized their specificity profiles using peptide phage-display libraries and scanning mutagenesis, which suggested a significantly enlarged recognition site of the high-specificity affinity clamps. The crystal structure of a high-specificity affinity clamp showed extensive contacts with a portion of the peptide ligand that is not recognized by the parent PDZ domain, thus rationalizing the improvement of the specificity of the affinity clamp. A comparison with another affinity clamp structure showed that, although both had extensive contacts between PDZ and FN3 domains, they exhibited a large offset in the relative position of the two domains. Our results indicate that linked domains could rapidly fuse and evolve as a single functional module, and that the inherent plasticity of domain interfaces allows for the generation of diverse active-site topography. These attributes of directed domain-interface evolution provide facile means to generate synthetic proteins with a broad range of functions.     

Characterization of a central Ca2+/calmodulin-dependent protein kinase IIalpha/beta binding domain in densin that selectively modulates glutamate receptor subunit phosphorylation

The densin C-terminal domain can target Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) in cells. Although the C-terminal domain selectively binds CaMKIIα in vitro, full-length densin associates with CaMKIIα or CaMKIIβ in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an "open" activated CaMKII conformation caused by Ca(2+)/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr-286/287 to Asp. Mutations in the densin-IN domain (L815E) or in the CaMKIIα/β catalytic domain (I205/206K) disrupt the interaction. The amino acid sequence of the densin-IN domain is similar to the CaMKII inhibitor protein, CaMKIIN, and a CaMKIIN peptide competitively blocks CaMKII binding to densin. CaMKII is inhibited by both CaMKIIN and the densin-IN domain, but the inhibition by densin is substrate-selective. Phosphorylation of a model peptide substrate, syntide-2, or of Ser-831 in AMPA receptor GluA1 subunits is fully inhibited by densin. However, CaMKII phosphorylation of Ser-1303 in NMDA receptor GluN2B subunits is not effectively inhibited by densin in vitro or in intact cells. Thus, densin can target multiple CaMKII isoforms to differentially modulate phosphorylation of physiologically relevant downstream targets.     

Solution structure of GOPC PDZ domain and its interaction with the C-terminal motif of neuroligin

GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein) represents a PDZ domain-containing protein associated with the Golgi apparatus, which plays important roles in vesicular trafficking in secretory and endocytic pathways. GOPC interacts with many other proteins, such as the Wnt receptors Frizzled 8 and neuroligin via its PDZ domain. Neuroligin is a neural cell-adhesion molecule of the post-synapse, which binds to the presynapse molecule neurexin to form a heterotypic intercellular junction. Here we report the solution structure of the GOPC PDZ domain by NMR. Our results show that it is a canonical class I PDZ domain, which contains two alpha-helices and six beta-strands. Using chemical shift perturbation experiments, we further studied the binding properties of the GOPC PDZ domain with the C-terminal motif of neuroligin. The observations showed that the ensemble of the interaction belongs to fast exchange with low affinity. The 3D model of the GOPC PDZ domain/neuroligin C-terminal peptide complex was constructed with the aid of the molecular dynamics simulation method. Our discoveries provide insight into the specific interaction of the GOPC PDZ domain with the C-terminal peptide of Nlg and also provide a general insight about the possible binding mode of the interaction of Nlg with other PDZ domain-containing proteins.     

Distinct binding specificity of the multiple PDZ domains of INADL, a human protein with homology to INAD from Drosophila melanogaster

PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different trans-membrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage lambda. We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster. This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position. Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference. However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide. Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures. For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain.     

Role of the PDZ domains in Escherichia coli DegP protein

PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The Escherichia coli essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.     

Bivalent peptides as PDZ domain ligands

A series of multivalent peptides, with the ability to simultaneously bind two separate PDZ domain proteins, has been designed, synthesized, and tested by isothermal titration calorimetry (ITC). The monomer sequences, linked with succinate, varied in length from five to nine residues. The thermodynamic binding parameters, in conjunction with results from mass spectrometry, indicate that a ternary complex is formed in which each peptide arm binds two equivalents of the third PDZ domain (PDZ3) of the neuronal protein PSD-95.     

Solution structure of the RIM1alpha PDZ domain in complex with an ELKS1b C-terminal peptide

PDZ domains are widespread protein modules that commonly recognize C-terminal sequences of target proteins and help to organize macromolecular signaling complexes. These sequences usually bind in an extended conformation to relatively shallow grooves formed between a beta-strand and an alpha-helix in the corresponding PDZ domains. Because of this binding mode, many PDZ domains recognize primarily the C-terminal and the antepenultimate side-chains of the target protein, which commonly conform to motifs that have been categorized into different classes. However, an increasing number of PDZ domains have been found to exhibit unusual specificities. These include the PDZ domain of RIMs, which are large multidomain proteins that regulate neurotransmitter release and help to organize presynaptic active zones. The RIM PDZ domain binds to the C-terminal sequence of ELKS with a unique specificity that involves each of the four ELKS C-terminal residues. To elucidate the structural basis for this specificity, we have determined the 3D structure in solution of an RIM/ELKS C-terminal peptide complex using NMR spectroscopy. The structure shows that the RIM PDZ domain contains an unusually deep and narrow peptide-binding groove with an exquisite shape complementarity to the four ELKS C-terminal residues in their bound conformation. This groove is formed, in part, by a set of side-chains that is conserved selectively in RIM PDZ domains and that hence determines, at least in part, their unique specificity.     

The ornithodorin-thrombin crystal structure,a key to the TAP enigma?

Ornithodorin, isolated from the blood sucking soft tick Ornithodoros moubata, is a potent (Ki = 10(-12) M) and highly selective thrombin inhibitor. Internal sequence homology indicates a two domain protein. Each domain resembles the Kunitz inhibitor basic pancreatic trypsin inhibitor (BPTI) and also the tick anticoagulant peptide (TAP) isolated from the same organism. The 3.1 A crystal structure of the ornithodorin-thrombin complex confirms that both domains of ornithodorin exhibit a distorted BPTI-like fold. The N-terminal portion and the C-terminal helix of each domain are structurally very similar to BPTI, whereas the regions corresponding to the binding loop of BPTI adopt different conformations. Neither of the two 'reactive site loops' of ornithodorin contacts the protease in the ornithodorin-thrombin complex. Instead, the N-terminal residues of ornithodorin bind to the active site of thrombin, reminiscent of the thrombin-hirudin interaction. The C-terminal domain binds at the fibrinogen recognition exosite. Molecular recognition of its target protease by this double-headed Kunitz-type inhibitor diverges considerably from other members of this intensely studied superfamily. The complex structure provides a model to explain the perplexing results of mutagenesis studies on the TAP-factor Xa interaction.     

C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease.

The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free alpha-carboxylate at the C-terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C-terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.     

Crystal structure of the Shank PDZ-ligand complex reveals a class I PDZ interaction and a novel PDZ-PDZ dimerization

The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinase-associated protein (GKAP1a) determined at 1.8- and 2.25-A resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at -1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at -3 and -5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved beta B/beta C loop and N-terminal beta A strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal beta A strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies.     

Regulation of Pin1 peptidyl-prolyl cis/trans isomerase activity by its WW binding module on a multi-phosphorylated peptide of Tau protein

The WW module of the peptidyl-prolyl cis/trans isomerase Pin1 targets specifically phosphorylated proteins involved in the cell cycle through the recognition of phospho-Thr(Ser)-Pro motifs. When the microtubule-associated Tau protein becomes hyperphosphorylated, it equally becomes a substrate for Pin1, with two recognition sites described around the phosphorylated Thr212 and Thr231. The Pin1 WW domain binds both sites with moderate affinity, but only the Thr212-Pro213 bond is isomerized by the catalytic domain of Pin1. We show here that, in a peptide carrying a single recognition site, the WW module increases significantly the enzymatic isomerase activity of Pin1. However, with addition of a second recognition motif, the affinity of both the WW and catalytic domain for the substrate increases, but the isomerization efficacy decreases. We therefore conclude that the WW domain can act as a negative regulator of enzymatic activity when multiple phosphorylation is present, thereby suggesting a subtle mechanism of its functional regulation.