The Experts below are selected from a list of 12513 Experts worldwide ranked by ideXlab platform
Mingjie Zhang - One of the best experts on this subject based on the ideXlab platform.
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redox regulated lipid membrane binding of the pick1 PDZ Domain
Biochemistry, 2010Co-Authors: Jiang Yu, Chong Shen, Mingjie ZhangAbstract:PICK1 is a PDZ/BAR Domain-containing scaffold protein that regulates the trafficking of many receptors and ion channels, including AMPA receptors. In addition to binding to a wide spectrum of target proteins to be transported, the PICK1 PDZ Domain, via its conserved CPC motif, has also been shown to bind to lipid membranes. However, the molecular basis of the CPC motif-mediated lipid membrane binding of the PICK1 PDZ Domain is not known. Here we show that the Cys residues in the CPC motif of the PICK1 PDZ Domain forms reversible, intermolecular disulfide bonds under mild oxidation conditions. Importantly, formation of the disulfide-mediated dimer abolishes the lipid membrane binding capacity of the PICK1 PDZ Domain and thereby is expected to alter the cellular functions of PICK1. The structures of the PDZ dimers provide atomic-scale pictures of disulfide-mediated PICK1 dimer formation and a molecular explanation of the oxidation-induced dissociation of PICK1 from membranes. We propose that the PICK1-media...
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organization and dynamics of PDZ Domain related supramodules in the postsynaptic density
Nature Reviews Neuroscience, 2009Co-Authors: Wei Feng, Mingjie ZhangAbstract:Catalytically inactive scaffold proteins are major constituents of the postsynaptic density. Feng and Zhang describe the distinct binding properties of multi-PDZ-Domain-containing scaffold proteins that enable them to actively participate in the dynamic regulation of signalling events at the synapse.
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clustering and synaptic targeting of pick1 requires direct interaction between the PDZ Domain and lipid membranes
The EMBO Journal, 2007Co-Authors: Hao Wu, Chong Shen, Mingjie ZhangAbstract:Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ Domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR Domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ Domain can directly interact with lipid membranes. The PDZ Domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved ‘Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ Domain, but also provided direct evidence supporting the functional relevance of the PDZ–lipid interaction.
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Synaptic PDZ Domain-mediated Protein Interactions Are Disrupted by Inhalational Anesthetics
Journal of Biological Chemistry, 2003Co-Authors: Ming Fang, Mingjie Zhang, Fahu He, Claire F. Levine, Chih Ling Chou, Scheherazade Sadegh-nasseri, Roger A. JohnsAbstract:Abstract Anesthetics exert multiple effects on the central nervous system through altering synaptic transmission, but the mechanisms for this process are poorly understood. PDZ Domain-mediated protein interactions play a central role in organizing signaling complexes around synaptic receptors for efficient signal transduction. We report here that clinically relevant concentrations of inhalational anesthetics dose-dependently and specifically inhibit the PDZ Domain-mediated protein interaction between PSD-95 or PSD-93 and the N-methyl-d-aspartate receptor or neuronal nitric-oxide synthase. These inhibitory effects are immediate, potent, and reversible and occur at a hydrophobic peptide-binding groove on the surface of the second PDZ Domain of PSD-95 in a manner relevant to anesthetic action. These findings reveal the PDZ Domain as a new molecular target for inhalational anesthetics.
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organization of signaling complexes by PDZ Domain scaffold proteins
Accounts of Chemical Research, 2003Co-Authors: Mingjie Zhang, Wenning WangAbstract:Transduction of biological signals from receptors at the plasma membrane to their targets in cytoplasm and nucleus relies on specific protein-protein interactions. A common strategy used by cells is to organize proteins in the same signaling cascade into large molecular weight, multiprotein complexes. PDZ Domain proteins have been shown to play important roles in assembling various signaling complexes. Here, we first present biophysical basis of the advantages of organizing proteins in a signaling cascade into a clustered multiprotein complex. We then discuss the structure, ligand binding, and function of PDZ Domains in organizing synaptic signaling complexes.
Richard L. Huganir - One of the best experts on this subject based on the ideXlab platform.
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potent PDZ Domain pick1 inhibitors that modulate amyloid beta mediated synaptic dysfunction
Scientific Reports, 2018Co-Authors: Laura Silvian, Douglas Marcotte, Charles C Banos, Timothy R Chan, Robert M Arduini, Darren P Baker, Chris Bergeron, Catherine Hession, Fang Qian, Richard L. HuganirAbstract:Protein interacting with C kinase (PICK1) is a scaffolding protein that is present in dendritic spines and interacts with a wide array of proteins through its PDZ Domain. The best understood function of PICK1 is regulation of trafficking of AMPA receptors at neuronal synapses via its specific interaction with the AMPA GluA2 subunit. Disrupting the PICK1-GluA2 interaction has been shown to alter synaptic plasticity, a molecular mechanism of learning and memory. Lack of potent, selective inhibitors of the PICK1 PDZ Domain has hindered efforts at exploring the PICK1-GluA2 interaction as a therapeutic target for neurological diseases. Here, we report the discovery of PICK1 small molecule inhibitors using a structure-based drug design strategy. The inhibitors stabilized surface GluA2, reduced Aβ-induced rise in intracellular calcium concentrations in cultured neurons, and blocked long term depression in brain slices. These findings demonstrate that it is possible to identify potent, selective PICK1-GluA2 inhibitors which may prove useful for treatment of neurodegenerative disorders.
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ribosomal s6 kinase 2 interacts with and phosphorylates PDZ Domain containing proteins and regulates ampa receptor transmission
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Gareth M Thomas, Gavin Rumbaugh, Dana B Harrar, Richard L. HuganirAbstract:Extracellular signal-regulated kinase (ERK) signaling is important for neuronal synaptic plasticity. We report here that the protein kinase ribosomal S6 kinase (RSK)2, a downstream target of ERK, uses a C-terminal motif to bind several PDZ Domain proteins in heterologous systems and in vivo. Different RSK isoforms display distinct specificities in their interactions with PDZ Domain proteins. Mutation of the RSK2 PDZ ligand does not inhibit RSK2 activation in intact cells or phosphorylation of peptide substrates by RSK2 in vitro but greatly reduces RSK2 phosphorylation of PDZ Domain proteins of the Shank family in heterologous cells. In primary neurons, NMDA receptor (NMDA-R) activation leads to ERK and RSK2 activation and RSK-dependent phosphorylation of transfected Shank3. RSK2–PDZ Domain interactions are functionally important for synaptic transmission because neurons expressing kinase-dead RSK2 display a dramatic reduction in frequency of AMPA-type glutamate receptor-mediated miniature excitatory postsynaptic currents, an effect dependent on the PDZ ligand. These results suggest that binding of RSK2 to PDZ Domain proteins and phosphorylation of these proteins or their binding partners regulates excitatory synaptic transmission.
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cerebellar long term depression requires pkc regulated interactions between glur2 3 and PDZ Domain containing proteins
Neuron, 2000Co-Authors: Hee Jung Chung, Richard L. Huganir, Cornelia Wihler, David J LindenAbstract:Abstract Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. Recently, it was shown that AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ Domain–containing proteins GRIP/ABP and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuated LTD induction by glutamate/depolarization pairing, as did antibodies directed against the PDZ Domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ Domain–containing proteins.
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phosphorylation of the ampa receptor subunit glur2 differentially regulates its interaction with PDZ Domain containing proteins
The Journal of Neuroscience, 2000Co-Authors: Hee Jung Chung, Robert H Scannevin, Xiaoqun Zhang, Richard L. HuganirAbstract:PSD-95, DLG, ZO-1 (PDZ) Domain-mediated protein interactions have been shown to play important roles in the regulation of glutamate receptor function at excitatory synapses. Recent studies demonstrating the rapid regulation of AMPA receptor function during synaptic plasticity have suggested that AMPA receptor interaction with PDZ Domain-containing proteins may be dynamically modulated. Here we show that PKC phosphorylation of the AMPA receptor GluR2 subunit differentially modulates its interaction with the PDZ Domain-containing proteins GRIP1 and PICK1. The serine residue [serine-880 (Ser880)] in the GluR2 C-terminal sequence (IESVKI) critical for PDZ Domain binding is a substrate of PKC and is phosphorylated in vivo . In vitro binding and coimmunoprecipitation studies show that phosphorylation of serine-880 within the GluR2 PDZ ligand significantly decreases GluR2 binding to GRIP1 but not to PICK1. Immunostaining of cultured hippocampal neurons demonstrates that the Ser880-phosphorylated GluR2 subunits are enriched and colocalized with PICK1 in the dendrites, with very little staining observed at excitatory synapses. Interestingly, PKC activation in neurons increases the Ser880 phosphorylation of GluR2 subunits and recruits PICK1 to excitatory synapses. Moreover, PKC stimulation in neurons results in rapid internalization of surface GluR2 subunits. These results suggest that GluR2 phosphorylation of serine-880 may be important in the regulation of the AMPA receptor internalization during synaptic plasticity.
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clustering of ampa receptors by the synaptic PDZ Domain containing protein pick1
Neuron, 1999Co-Authors: Xiaoqun Zhang, Jeff Staudinger, Richard L. HuganirAbstract:Abstract Synaptic clustering of neurotransmitter receptors is crucial for efficient signal transduction and integration in neurons. PDZ Domain–containing proteins such as PSD-95/SAP90 interact with the intracellular C termini of a variety of receptors and are thought to be important in the targeting and anchoring of receptors to specific synapses. Here, we show that PICK1 (protein interacting with C kinase), a PDZ Domain–containing protein, interacts with the C termini of α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in vitro and in vivo. In neurons, PICK1 specifically colocalizes with AMPA receptors at excitatory synapses. Furthermore, PICK1 induces clustering of AMPA receptors in heterologous expression systems. These results suggest that PICK1 may play an important role in the modulation of synaptic transmission by regulating the synaptic targeting of AMPA receptors.
Ernesto J. Fuentes - One of the best experts on this subject based on the ideXlab platform.
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Emerging Themes in PDZ Domain Signaling: Structure, Function, and Inhibition.
International Review of Cell and Molecular Biology, 2018Co-Authors: Ernesto J. FuentesAbstract:Abstract Post-synaptic density-95, disks-large and zonula occludens-1 (PDZ) Domains are small globular protein–protein interaction Domains widely conserved from yeast to humans. They are composed of ∼90 amino acids and form a classical two α-helical/six β-strand structure. The prototypical ligand is the C-terminus of partner proteins; however, they also bind internal peptide sequences. Recent findings indicate that PDZ Domains also bind phosphatidylinositides and cholesterol. Through their ligand interactions, PDZ Domain proteins are critical for cellular trafficking and the surface retention of various ion channels. In addition, PDZ proteins are essential for neuronal signaling, memory, and learning. PDZ proteins also contribute to cytoskeletal dynamics by mediating interactions critical for maintaining cell–cell junctions, cell polarity, and cell migration. Given their important biological roles, it is not surprising that their dysfunction can lead to multiple disease states. As such, PDZ Domain–containing proteins have emerged as potential targets for the development of small molecular inhibitors as therapeutic agents. Recent data suggest that the critical binding function of PDZ Domains in cell signaling is more than just glue, and their binding function can be regulated by phosphorylation or allosterically by other binding partners. These studies also provide a wealth of structural and biophysical data that are beginning to reveal the physical features that endow this small modular Domain with a central role in cell signaling.
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Thermodynamic and Dynamic Basis for the Broadened Ligand Specificity of a Tiam2 PDZ Domain Mutant
Biophysical Journal, 2015Co-Authors: Ernesto J. Fuentes, Lisa C. Golden, Liping YuAbstract:PDZ (PSD-95/Dlg/ZO-1) Domains are protein-protein interaction modules that typically recognize their binding partners through the use of two specificity pockets. Here we examine the consequence of mutating four residues in the Tiam2 PDZ Domain specificity pockets to produce a quadruple mutant (QM). Equilibrium binding studies show that the specificity of the Tiam2 QM mutant is similar to that seen in the wild type Tiam1 PDZ Domain. Isothermal titration calorimetry experiments show a larger entropic contribution to ligand binding in the QM PDZ Domain compared to the WT PDZ Domain. Double-mutant cycle analysis uncovered cooperativity between residues in the two specificity pockets with respect to both ligand binding and protein folding. NMR-based HSCQ studies reveal that the wild type Tiam2 PDZ has severe line broadening in several loop regions, while the QM PDZ had additional regions of line broadening. However, peptide ligand binding dampens line broadening for both the Tiam2 WT and QM PDZ Domains. Finally, CPMG dispersion experiments indicate that the number of residues experiencing micro to millisecond motions is significantly increased in the QM PDZ Domain. We propose a model where enhanced dynamics alters the QM PDZ Domain conformational ensemble allowing for broader ligand specificity relative to the WT PDZ Domain.
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Specificity, Structure and Dynamics of Tiam1 PDZ Domain Ligand-Bound Complexes
Biophysical Journal, 2012Co-Authors: Ernesto J. Fuentes, Tyson R. Shepherd, David C. Speckhard, Ann M. MurrayAbstract:PSD-95/DlgA/ZO-1 (PDZ) Domains are among the most abundant protein-protein interaction Domains in the human proteome and typically bind the 4-10 C-terminal residues of its interaction partner with exquisite specificity. We used two homologous PDZ Domains from the Tiam-family of guanine nucleotide exchange factors to investigate PDZ specificity. The Tiam1 and Tiam2 PDZ Domains have overlapping but distinct ligand binding specificity, and this is exemplified by their unique preferences for C-terminal peptides derived from the syndecan1, Caspr4 and neurexin1 adhesion proteins. The Tiam1 PDZ Domain binds syndecan1 and Caspr4 but not neurexin1, while the Tiam2 PDZ Domain binds Caspr4 and neurexin1 but not syndecan1. Amino acid sequence comparison of Tiam-family PDZ Domains revealed that four residues critical for ligand specificity are not conserved. Remarkably, substitution of these four residues in the Tiam1 PDZ for those found in the Tiam2 PDZ Domain switched ligand specificity. To understand the structural and dynamic basis for this change in specificity we used X-ray crystallography and solution NMR methods, respectively. We determined the crystal structures of wild type Tiam1 PDZ Domain bound to syndecan1 and phosphorylated syndecan1 peptides and the Tiam1 PDZ quadruple mutant (QM) bound to Caspr4 and neurexin1 peptides. Comparison of the crystal structures of the Tiam1 PDZ-syndecan1 and PDZ-phosphorylated syndecan1 showed that a distinct specificity pocket is used to accommodate the phosphoryl group. The crystal structure of the Tiam1 QM PDZ Domain showed a unique side chain stacking interaction between aromatic residues in the PDZ Domain and the Caspr4 ligand. Side chain methyl relaxation experiments revealed distinct patterns of dynamics in the Tiam1 PDZ-syndecan1 and PDZ-Caspr4 complexes. Collectively, the structures and dynamics of physiologically-based PDZ Domain complexes are contributing to understanding the origin of PDZ specificity and function of Tiam-family PDZ Domains.
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The Tiam1 PDZ Domain couples to Syndecan1 and promotes cell-matrix adhesion.
Journal of Molecular Biology, 2010Co-Authors: Tyson R. Shepherd, Suzi M. Klaus, S. Ramaswamy, Kris A. Demali, Ernesto J. FuentesAbstract:The T-cell lymphoma invasion and metastasis gene 1 (Tiam1) is a guanine exchange factor (GEF) for the Rho-family GTPase Rac1 that is crucial for the integrity of adherens junctions, tight junctions, and cell-matrix interactions. This GEF contains several protein-protein interaction Domains, including a PDZ Domain. Earlier studies identified a consensus PDZ-binding motif and a synthetic peptide capable of binding to the Tiam1 PDZ Domain, but little is known about its ligand specificity and physiological role in cells. Here, we investigated the structure, specificity, and function of the Tiam1 PDZ Domain. We determined the crystal structures of the Tiam1 PDZ Domain free and in complex with a "model" peptide, which revealed the structural basis for ligand specificity. Protein database searches using the consensus PDZ-binding motif identified two eukaryotic cell adhesion proteins, Syndecan1 and Caspr4, as potential Tiam1 PDZ Domain binding proteins. Equilibrium binding experiments confirmed that C-terminal peptides derived from Syndecan1 and Caspr4 bound the Tiam1 PDZ Domain. NMR chemical shift perturbation experiments indicated that the Tiam1 PDZ/Syndecan1 and PDZ/Caspr4 complexes were structurally distinct and identified key residues likely to be responsible for ligand selectivity. Moreover, cell biological analysis established that Syndecan1 is a physiological binding partner of Tiam1 and that the PDZ Domain has a function in cell-matrix adhesion and cell migration. Collectively, our data provide insight into the structure, specificity, and function of the Tiam1 PDZ Domain. Importantly, our data report on a physiological role for the Tiam1 PDZ Domain and establish a novel link between two previously unrelated signal transduction pathways, both of which are implicated in cancer.
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hidden dynamic allostery in a PDZ Domain
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Chad M Petit, Jun Zhang, Paul J Sapienza, Ernesto J. FuentesAbstract:Abstract Structure–function relationships in proteins are predicated on the spatial proximity of noncovalently interacting groups of atoms. Thus, structural elements located away from a protein's active site are typically presumed to serve a stabilizing or scaffolding role for the larger structure. Here we report a functional role for a distal structural element in a PDZ Domain, even though it is not required to maintain PDZ structure. The third PDZ Domain from PSD-95/SAP90 (PDZ3) has an unusual additional third alpha helix (α3) that packs in contiguous fashion against the globular Domain. Although α3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of α3 reduces ligand affinity by 21-fold. Further investigation revealed that the difference in binding free energies between the full-length and truncated constructs is predominantly entropic in nature and that without α3, picosecond-nanosecond side-chain dynamics are enhanced throughout the Domain, as determined by 2H methyl NMR relaxation. Thus, the distal modulation of binding function appears to occur via a delocalized conformational entropy mechanism. Without removal of α3 and characterization of side-chain dynamics, this dynamic allostery would have gone unnoticed. Moreover, what appeared at first to be an artificial modification of PDZ3 has been corroborated by experimentally verified phosphorylation of α3, revealing a tangible biological mechanism for this novel regulatory scheme. This hidden dynamic allostery raises the possibility of as-yet unidentified or untapped allosteric regulation in this PDZ Domain and is a very clear example of function arising from dynamics rather than from structure.
Gary D Bader - One of the best experts on this subject based on the ideXlab platform.
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Proteome scanning to predict PDZ Domain interactions using support vector machines.
BMC Bioinformatics, 2010Co-Authors: Gary D BaderAbstract:Background PDZ Domains mediate protein-protein interactions involved in important biological processes through the recognition of short linear motifs in their target proteins. Two recent independent studies have used protein microarray or phage display technology to detect PDZ Domain interactions with peptide ligands on a large scale. Several computational predictors of PDZ Domain interactions have been developed, however they are trained using only protein microarray data and focus on limited subsets of PDZ Domains. An accurate predictor of genomic PDZ Domain interactions would allow the proteomes of organisms to be scanned for potential binders. Such an application would require an accurate and precise predictor to avoid generating too many false positive hits given the large amount of possible interactors in a given proteome. Once validated these predictions will help to increase the coverage of current PDZ Domain interaction networks and further our understanding of the roles that PDZ Domains play in a variety of biological processes.
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Proteome scanning to predict PDZ Domain interactions using support vector machines
BMC Bioinformatics, 2010Co-Authors: Shirley Hui, Gary D BaderAbstract:Background: PDZ Domains mediate protein-protein interactions involved in important biological processes through the recognition of short linear motifs in their target proteins. Two recent independent studies have used protein microarray or phage display technology to detect PDZ Domain interactions with peptide ligands on a large scale. Several computational predictors of PDZ Domain interactions have been developed, however they are trained using only protein microarray data and focus on limited subsets of PDZ Domains. An accurate predictor of genomic PDZ Domain interactions would allow the proteomes of organisms to be scanned for potential binders. Such an application would require an accurate and precise predictor to avoid generating too many false positive hits given the large amount of possible interactors in a given proteome. Once validated these predictions will help to increase the coverage of current PDZ Domain interaction networks and further our understanding of the roles that PDZ Domains play in a variety of biological processes.Results: We developed a PDZ Domain interaction predictor using a support vector machine (SVM) trained with both protein microarray and phage display data. In order to use the phage display data for training, which only contains positive interactions, we developed a method to generate artificial negative interactions. Using cross-validation and a series of independent tests, we showed that our SVM successfully predicts interactions in different organisms. We then used the SVM to scan the proteomes of human, worm and fly to predict binders for several PDZ Domains. Predictions were validated using known genomic interactions and published protein microarray experiments. Based on our results, new protein interactions potentially associated with Usher and Bardet-Biedl syndromes were predicted. A comparison of performance measures (F1 measure and FPR) for the SVM and published predictors demonstrated our SVM's improved accuracy and precision at proteome scanning.Conclusions: We built an SVM using mouse and human experimental training data to predict PDZ Domain interactions. We showed that it correctly predicts known interactions from proteomes of different organisms and is more accurate and precise at proteome scanning compared with published state-of-the-art predictors. ? 2010 Hui and Bader; licensee BioMed Central Ltd.
Sachdev S Sidhu - One of the best experts on this subject based on the ideXlab platform.
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Origins of PDZ Domain Ligand Specificity
2020Co-Authors: Nicholas J Skelton, Maria Teresa Pisabarro, Laurence A. Lasky, Michael F. T. Koehler, Kerry Zobel, Wai Lee Wong, Sachdev S SidhuAbstract:The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ Domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ Domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW 4 ETW 1 V; IC50 0.15 M) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp 1 are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ Domains), whereas the 2-3 loop provides a binding site for the Trp 4 side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ Domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ Domains present in the human genome.
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A structural portrait of the PDZ Domain family
Journal of Molecular Biology, 2014Co-Authors: Andreas Ernst, Brent A. Appleton, Christian Wiesmann, Yingnan Zhang, Ylva Ivarsson, David Gfeller, Sachdev S SidhuAbstract:PDZ (PSD-95/Discs-large/ZO1) Domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ Domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ Domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ Domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ Domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ Domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical Domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ Domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.
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Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ Domain.
Protein Science, 2007Co-Authors: Yingnan Zhang, Brent A. Appleton, Christian Wiesmann, Ping Wu, Sachdev S SidhuAbstract: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.
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comparative structural analysis of the erbin PDZ Domain and the first PDZ Domain of zo 1 insights into determinants of PDZ Domain specificity
Journal of Biological Chemistry, 2006Co-Authors: Brent A. Appleton, Yingnan Zhang, Sachdev S Sidhu, Ping Wu, Walter Hunziker, Nicholas J Skelton, Christian WiesmannAbstract:Abstract We report a structural comparison of the first PDZ Domain of ZO-1 (ZO1-PDZ1) and the PDZ Domain of Erbin (Erbin-PDZ). Although the binding profile of Erbin-PDZ is extremely specific ([D/E][T/S]WVCOOH), that of ZO1-PDZ1 is similar ([R/K/S/T][T/S][W/Y][V/I/L]COOH) but broadened by increased promiscuity for three of the last four ligand residues. Consequently, the biological function of ZO-1 is also broadened, as it interacts with both tight and adherens junction proteins, whereas Erbin is restricted to adherens junctions. Structural analyses reveal that the differences in specificity can be accounted for by two key differences in primary sequence. A reduction in the size of the hydrophobic residue at the base of the site0 pocket enables ZO1-PDZ1 to accommodate larger C-terminal residues. A single additional difference alters the specificity of both site-1 and site-3. In ZO1-PDZ1, an Asp residue makes favorable interactions with both Tyr-1 and Lys/Arg-3. In contrast, Erbin-PDZ contains an Arg at the equivalent position, and this side chain cannot accommodate either Tyr-1 or Lys/Arg-3 but, instead, interacts favorably with Glu/Asp-3. We propose a model for ligand recognition that accounts for interactions extending across the entire binding site but that highlights several key specificity switches within the PDZ Domain fold.
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origins of PDZ Domain ligand specificity structure determination and mutagenesis of the erbin PDZ Domain
Journal of Biological Chemistry, 2003Co-Authors: Nicholas J Skelton, Laurence A. Lasky, Michael F. T. Koehler, Kerry Zobel, Wai Lee Wong, Theresa M Pisabarro, Sachdev S SidhuAbstract:Abstract The LAP (leucine-rich repeatand PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ Domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ Domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW−4ETW−1V; IC50 = ∼0.15 μm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp−1 are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ Domains), whereas the β2-β3 loop provides a binding site for the Trp−4 side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ Domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ Domains present in the human genome.