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.     

Ligand-dependent dynamics and intramolecular signaling in a PDZ domain

Allosteric communication is a fundamental process that proteins use to propagate signals from one site to functionally important distal sites. Although allostery is usually associated with multimeric proteins and enzymes, “long-range” communication may be a fundamental property of proteins. In some cases, communication occurs with minimal structural change. PDZ (post-synaptic density-95/discs large/zonula occludens-1) domains are small, protein-protein binding modules that can use multiple surfaces for docking diverse molecules. Furthermore, these domains have long-range energetic couplings that link the ligand-binding site to distal regions of the structure. Here, we show that allosteric behavior in a representative member of the PDZ domain family may be directly detected using side-chain methyl dynamics measurements. The changes in side-chain dynamics parameters in the second PDZ domain from the human tyrosine phosphatase 1E (hPTP1E) were determined upon binding a peptide target. Long-range dynamic effects were detected that correspond to previously observed pair-wise energetic couplings. These results provide one of the first experimental examples for the potential role of ps-ns timescale dynamics in propagating long-range signals within a protein, and reinforce the idea that dynamic fluctuations in proteins contribute to allosteric signal transduction.     

Structural insight into the interaction between the p55 PDZ domain and glycophorin C

p55, a member of the membrane-associated guanylate kinase family, includes a PDZ domain that specifically interacts with the C-terminal region of glycophorin C in the ternary complex of p55, protein 4.1 and glycophorin C. Here we present the first NMR-derived complex structure of the p55 PDZ domain and the C-terminal peptide of glycophorin C, obtained by using a threonine to cysteine (T85C) mutant of the p55 PDZ domain and a phenylalanine to cysteine (F127C) mutant of the glycophorin C peptide. Our NMR results revealed that the two designed mutant molecules retain the specific interaction manner that exists between the wild type molecules and can facilitate the structure determination by NMR, due to the stable complex formation via an intermolecular disulfide bond. The complex structure provides insight into the specific interaction of the p55 PDZ domain with the two key residues, Ile128 and Tyr126, of glycophorin C.     

Zinc binding site in PICK1 is dominantly located at the CPC motif of its PDZ domain

PICK1 ( p rotein i nteracting with C k inase 1) containing a PDZ domain, a BAR domain, and two short acidic regions is as an adaptor protein that plays an important role in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor trafficking, cell morphology and migration, as well as in some diseases such as cancer, schizophrenia and pain. To better understand the physiological function of PICK1, we expressed the recombinant PICK1 and its truncated mutants in E.coli, and measured their zinc binding properties by fluorescence and competition assay. It is shown that PICK1 has one Zn²⁺-binding site. The Zn²⁺-binding properties of PICK1 are not appreciably affected after the removal of BARC domain (involving BAR domain and C-terminal acidic region). Deleting the N-terminal acidic region of NPDZ domain (involving PDZ domain and N-terminal acidic region) in PICK1 impairs its Zn²⁺-binding capacity.The mutation of the CPC (Cys-Pro-Cys) motif in the PDZ domain of PICK1 abolishes the ability of Zn²⁺-binding. In addition, Zn²⁺ can enhance the lipid-binding ability of PDZ domain as observed in both protein-lipid overlay assay and fluorescence analysis. The results presented in this report suggested that Zn²⁺ plays a regulatory role in the trafficking of PICK1 from the cytoplasm to cell membrane.     

NMR evaluation of interactions between substituted-indole and PDZ1 domain of PSD-95

We synthesized small organic molecules designed as PDZ ligands. These indole-based compounds were evaluated for their interaction with the PDZ1 domain of the post-synaptic density 95 (PSD-95) protein. Three molecules were found to interact with the targeted PDZ protein by NMR. One of them showed chemical shift perturbations closely related to the natural ligands.     

Lock and chop: A novel method for the generation of a PICK1 PDZ domain and piperidine‐based inhibitor co‐crystal structure

The membrane protein interacting with kinase C1 (PICK1) plays a trafficking role in the internalization of neuron receptors such as the amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA) receptor. Reduction of surface AMPA type receptors on neurons reduces synaptic communication leading to cognitive impairment in progressive neurodegenerative diseases such as Alzheimer disease. The internalization of AMPA receptors is mediated by the PDZ domain of PICK1 which binds to the GluA2 subunit of AMPA receptors and targets the receptor for internalization through endocytosis, reducing synaptic communication. We planned to block the PICK1‐GluA2 protein–protein interaction with a small molecule inhibitor to stabilize surface AMPA receptors as a therapeutic possibility for neurodegenerative diseases. Using a fluorescence polarization assay, we identified compound BIO124 as a modest inhibitor of the PICK1‐GluA2 interaction. We further tried to improve the binding affinity of BIO124 using structure‐aided drug design but were unsuccessful in producing a co‐crystal structure using previously reported crystallography methods for PICK1. Here, we present a novel method through which we generated a co‐crystal structure of the PDZ domain of PICK1 bound to BIO124.     

Simulations of a specific inhibitor of the dishevelled PDZ domain

The dishevelled (Dvl) PDZ domain is believed to play an essential role in the canonical and noncanonical Wnt signaling pathways, which are involved in embryo development as well as in tumorigenesis. Also, it binds directly to frizzled (Fz) receptors. An organic molecule (NSC668036) from the National Cancer Institute small-molecule library has been identified to be able to bind to the Dvl PDZ domain. Molecular dynamics simulation was used to provide detailed analyses of the binding between them. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Solution structure of the PDZ2 domain from cytosolic human phosphatase hPTP1E complexed with a peptide reveals contribution of the beta2-beta3 loop to PDZ domain-ligand interactions

The solution structure of the second PDZ domain from human phosphatase hPTP1E in complex with a C-terminal peptide from the guanine nucleotide exchange factor RA-GEF-2 has been determined using 2D and 3D heteronuclear NMR experiments. Compared to previously solved structures, the hPTP1E complex shows an enlarged interaction surface with the C terminus of the bound peptide. Novel contacts were found between the long structured beta2/beta3 loop of the PDZ domain and the sixth amino acid residue from the C terminus of the peptide. This work underlines the importance of the beta2/beta3 loop for ligand selection by PDZ domains.     

Contrasting roles of dynamics in protein allostery: NMR and structural studies of CheY and the third PDZ domain from PSD-95

Allosteric regulation is a ubiquitous phenomenon exploited in biological processes to control cells in a myriad of ways. It is also of emerging interest in the design of functional proteins and therapeutics. Even though allostery was proposed over 50 years ago and has been studied intensively from a structural perspective, many key details of allosteric mechanisms remain mysterious. Over the last decade significant attention has been paid to the “dynamic component” of allostery, as opposed to the analysis of rigid structures. Nuclear magnetic resonance spectroscopy and its ability to detect conformationally dynamic processes at atomic resolution have played an important role in expanding our understanding of allosteric mechanisms and opening up new questions. This article focuses on work that highlights how protein dynamics can factor into allosteric processes in distinct ways. Two cases are contrasted. The first considers the “traditionally allosteric” protein CheY, which undergoes a conformational change as a key element of its allostery. The second considers the more rarely observed “dynamic allostery” in a PDZ domain, in which allosteric behavior arises from changes in internal structural dynamics. Interestingly, the dynamic processes in these two contrasting examples occur on different timescales. In the case of the PDZ domain, subsequent experimental and computational work is reviewed to reveal a more complete picture of this interesting case of allostery.     

Using a collection of MUPP1 domains to investigate the similarities of neurotransmitter transporters C-terminal PDZ motifs

A ubiquitous feature of neurotransmitter transporters is the presence of short C-terminal PDZ binding motifs acting as important trafficking elements. Depending on their very C-terminal sequences, PDZ binding motifs are usually divided into at least three groups; however this classification has recently been questioned. To introduce a 3D aspect into transporter’s PDZ motif similarities, we compared their interactions with the natural collection of all 13 PDZ domains of the largest PDZ binding protein MUPP1. The GABA, glycine and serotonin transporters showed unique binding preferences scattered over one or several MUPP1 domains. On the contrary, the dopamine and norepinephrine transporter PDZ motifs did not show any significant affinity to MUPP1 domains. Interestingly, despite their terminal sequence diversity all three GABA transporter PDZ motifs interacted with MUPP1 domain 7. These results indicate that similarities in binding schemes of individual transporter groups might exist. Results also suggest the existence of variable PDZ binding modes, allowing several transporters to interact with identical PDZ domains and potentially share interaction partners in vivo.     

Structure determination and ligand interactions of the PDZ2b domain of PTP-Bas (hPTP1E): splicing-induced modulation of ligand specificity

Two versions of the PDZ2 domain of the protein tyrosine phosphatase PTP-Bas/human PTP-BL are generated by alternative splicing. The domains differ by the insertion of five amino acid residues and their affinity to the tumour suppressor protein APC. Whereas PDZ2a is able to bind APC in the nanomolar range, PDZ2b shows no apparent interaction with APC. Here the solution structure of the splicing variant of PDZ2 with the insertion has been determined using 2D and 3D heteronuclear NMR experiments. The structural reason for the changed binding specificity is the reorientation of the loop with extra five amino acid residues, which folds back onto beta-strands two and three. In addition the side-chain of Lys32 closes the binding site of the APC binding protein and the two helices, especially alpha-helix 2, change their relative position to the protein core. Consecutively, the binding site is sterically no longer fully accessible. From the NMR-titration studies with a C-terminal APC-peptide the affinity of the peptide with the protein can be estimated as 540(+/-40)microM. The binding site encompasses part of the analogous binding site of PDZ2a as already described previously, yet specific interaction sites are abolished by the insertion of amino acids in PDZ2b. As shown by high-affinity chromatography, GST-PDZ2b and GST-PDZ2a bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles with a dissociation constant K(D) of 21 microM and 55 microM, respectively. In line with these data PDZ2b binds isolated, dissolved PIP(2) and PIP(3) (phosphatidylinositol 3,4,5-trisphosphate) molecules specifically with a lower K(D) of 230(+/-20)microM as detected by NMR spectroscopy. The binding site could be located by our studies and involves the residues Ile24, Val26, Val70, Asn71, Gly77, Ala78, Glu85, Arg88, Gly91 and Gln92. PIP(2) and PIP(3) binding takes place in the groove of the PDZ domain that is normally part of the APC binding site.     

Extensions of PDZ domains as important structural and functional elements

‘Divide and conquer’ has been the guiding strategy for the study of protein structure and function. Proteins are divided into domains with each domain having a canonical structural definition depending on its type. In this review, we push forward with the interesting observation that many domains have regions outside of their canonical definition that affect their structure and function; we call these regions ‘extensions’. We focus on the highly abundant PDZ (PSD-95, DLG1 and ZO-1) domain. Using bioinformatics, we find that many PDZ domains have potential extensions and we developed an openly-accessible website to display our results (http://bcz102.ust.hk/pdzex/). We propose, using well-studied PDZ domains as illustrative examples, that the roles of PDZ extensions can be classified into at least four categories: 1) protein dynamics-based modulation of target binding affinity, 2) provision of binding sites for macro-molecular assembly, 3) structural integration of multi-domain modules, and 4) expansion of the target ligand-binding pocket. Our review highlights the potential structural and functional importance of domain extensions, highlighting the significance of looking beyond the canonical boundaries of protein domains in general.     

Catalytic domain of MMP20 (Enamelysin) - the NMR structure of a new matrix metalloproteinase

The solution structure of the catalytic domain of MMP-20, a member of the matrix metalloproteinases family not yet structurally characterized, complexed with N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH), is here reported and compared with other MMPs-NNGH adducts. The backbone dynamic has been characterized as well. We have found that, despite the same fold and very high overall similarity, the present structure experiences specific structural and dynamical similarities with some MMPs and differences with others, around the catalytic cavity. The present solution structure, not only contributes to fill the gap of structural knowledge on human MMPs, but also provides further information to design more selective and efficient inhibitors for a specific member of this class of proteins.     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P⁻³, which means that the P⁻³ residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins,and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class Ⅱ PDZ motif, it also binds to Class Ⅰ and Class Ⅲ motifs, and exhibits restricted variability at P-3, which means that the P-3 residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi.Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

A novel Wnt5a‐Frizzled4 signaling pathway mediates activity‐independent dendrite morphogenesis via the distal PDZ motif of Frizzled 4

The morphology of the dendritic tree is critical to neuronal function and neural circuit wiring. Several Wnt family members have been demonstrated to play important roles in dendrite development. However, the Wnt receptors responsible for mediating this process remain largely elusive. Using primary hippocampal neuronal cultures as a model system, we report that Frizzled4 (Fzd4), a member of the Fzd family of Wnt receptors, specifically signals downstream of Wnt5a to promote dendrite branching and growth. Interestingly, the less conserved distal PDZ binding motif of Fzd4, and not its conserved proximal Dvl‐interacting PDZ motif, is required for mediating this effect. We further showed that Dvl signaled parallel to and independent of Fzd4 in promoting dendrite growth. Unlike most previously described pathways, Wnt5a/Fzd4 signaling promoted dendrite development in an activity‐independent and autocrine fashion. Together, these results provide the first identification of a Wnt receptor for regulating dendrite development in the mammalian system, and demonstrate a novel function of the distal PDZ motif of Fzd4 in dendrite morphogenesis, thereby expanding our knowledge of the complex roles of Wnt signaling in neural development. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 805℃822, 2015     

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.     

Solution structure and backbone dynamics of the AF-6 PDZ domain/Bcr peptide complex

The human AF-6, a scaffold protein between cell membrane-associated proteins and the actin cytoskeleton, plays an important role in special cell-cell junctions and signal transduction. It can be phosphorylated by the protein kinase Bcr, which allows efficient binding of the C terminus of Bcr to the PDZ domain of AF-6 and consequently enhances the binding affinity of AF-6 to Ras. Formation of the AF-6, Bcr, and Ras ternary complex results in down-regulation of the Ras-mediated signal transduction pathway. To better understand the molecular basis for the recognition of the AF-6 PDZ domain and Bcr, we solve the solution structure of the AF-6 PDZ domain complexed with the C-terminal peptide of Bcr and explore the interactions between them in detail. Compared with previously reported structures, the complex exhibits a noncanonical binding mode of PDZ/peptide. Owing to the distinct residues involved in the AF-6 PDZ domain and Bcr peptide interaction, the interaction mode does not adapt to the existing classification rules that have been put forward, based on the ligand or the PDZ domain specificity. Furthermore, the PDZ domain of AF-6 can bind to the C terminus of Bcr efficiently after phosphorylation of AF-6 by the Bcr kinase. The phosphorylation may induce a conformational change of AF-6, which makes the binding surface on the PDZ domain accessible to Bcr for efficient binding. This study not only characterizes the structural details of the AF-6 PDZ/Bcr peptide complex, but also provides a potential target for future drug design and disease therapy.     

Identification of small-molecule compounds targeting the dishevelled PDZ domain by virtual screening and binding studies

The Dishevelled (Dvl) protein, which conveys signals from receptors to the downstream effectors, is a critical constituent of the Wnt/β-catenin signaling pathway. Because the PDZ domain of Dvl protein functions through associations with a wide range of protein partners, Dvl protein involved in the Wnt signaling pathway has been considered to be therapeutic targets in cancers. In this study, we performed structure-based pharmacophore model of the Dvl PDZ domain to discover novel small-molecule binders and identified eight compounds with micromolar affinity. The most potent compound identified, BMD4702, efficiently bound to the Dvl PDZ domain with 11.2 μM affinity and had a 0.186 μM K_D value according to surface plasmon resonance and fluorescence spectroscopy, respectively. Combining both structural–kinetic relationship analyses and docking studies, we fourmulated that the ligand-binding site is composed of three H-bonds and three hydrophobic features. Thus, our approach led to the identification of potent binders of the Dvl PDZ domain and the findings provide novel insights into structure-based approaches to design high-affinity binders for the Dvl PDZ domain.     

Survey on the PABC recognition motif PAM2

The PABP-interacting motif PAM2 has been identified in various eukaryotic proteins as an important binding site for the PABC domain. This domain is contained in homologs of the poly(A)-binding protein PABP and the ubiquitin-protein ligase HYD. Despite the importance of the PAM2 motif, a comprehensive analysis of its occurrence in different proteins has been missing. Using iterated sequence profile searches, we obtained an extensive list of proteins carrying the PAM2 motif. We discuss their functional context and domain architecture, which often consists of RNA-binding domains. Our list of PAM2 motif proteins includes eukaryotic homologs of eRF3/GSPT1/2, PAIP1/2, Tob1/2, Ataxin-2, RBP37, RBP1, Blackjack, HELZ, TPRD, USP10, ERD15, C1D4.14, and the viral protease P29. The identification of the PAM2 motif in as yet uncharacterized proteins can give valuable hints with respect to their cellular function and potential interaction partners and suggests further experimentation. It is also striking that the PAM2 motif appears to occur solely outside globular protein domains.