scholarly journals Interactomic affinity profiling by holdup assay: acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

2020 ◽  
Author(s):  
Pau Jané ◽  
Gergő Gógl ◽  
Camille Kostmann ◽  
Goran Bich ◽  
Virginie Girault ◽  
...  
Keyword(s):  
High Throughput ◽  
Experimental Approach ◽  
Consensus Sequence ◽  
Pdz Domain ◽  
Binding Motif ◽  
Pdz Domains ◽  
Post Translational Modifications ◽  
Binding Profile ◽  
Protein Motifs ◽  
The Impact

AbstractProtein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and fluorescence polarization to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN phosphatase towards the 266 known human PDZ domains. Inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile of the PTEN PBM. A specificity index is also introduced to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.

scholarly journals Interactomic affinity profiling by holdup assay: Acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244613
Author(s):  
Pau Jané ◽  
Gergő Gógl ◽  
Camille Kostmann ◽  
Goran Bich ◽  
Virginie Girault ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Pdz Domain ◽  
Pi3k Pathway ◽  
Binding Motif ◽  
Pdz Domains ◽  
Post Translational Modifications ◽  
Binding Profile ◽  
Protein Motifs ◽  
C Terminus ◽  
The Impact

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. PTEN, a lipid phosphatase involved in phosphatidylinositol 3-kinase (PI3K) pathway, contains such a short motif located at the extreme C-terminus capable to recognize PDZ domains. It has been shown that the acetylation of this motif could modulate the interaction with several PDZ domains. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and competitive fluorescence polarization technique to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN. We substantially extended the previous knowledge towards the 266 known human PDZ domains, generating the full PDZome-binding profile of the PTEN PBM. We confirmed that inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile. A numerical specificity index is also introduced as an attempt to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.

scholarly journals Design of a companion bioinformatic tool to detect the emergence and geographical distribution of SARS-CoV-2 Spike protein genetic variants

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Alice Massacci ◽  
Eleonora Sperandio ◽  
Lorenzo D’Ambrosio ◽  
Mariano Maffei ◽  
Fabio Palombo ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Consensus Sequence ◽  
Cell Epitope ◽  
Vaccine Design ◽  
Binding Domain ◽  
Spike Protein ◽  
Antibody Activity ◽  
Binding Motif ◽  
Receptor Binding Domain ◽  
The Impact

Abstract Background Tracking the genetic variability of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a crucial challenge. Mainly to identify target sequences in order to generate robust vaccines and neutralizing monoclonal antibodies, but also to track viral genetic temporal and geographic evolution and to mine for variants associated with reduced or increased disease severity. Several online tools and bioinformatic phylogenetic analyses have been released, but the main interest lies in the Spike protein, which is the pivotal element of current vaccine design, and in the Receptor Binding Domain, that accounts for most of the neutralizing the antibody activity. Methods Here, we present an open-source bioinformatic protocol, and a web portal focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Furthermore, we provide immunogenomic analyses to understand the impact of the most frequent RBD variations. Results Results on the whole GISAID sequence dataset at the time of the writing (October 2020) reveals an emerging mutation, S477N, located on the central part of the Spike protein Receptor Binding Domain, the Receptor Binding Motif. Immunogenomic analyses revealed some variation in mutated epitope MHC compatibility, T-cell recognition, and B-cell epitope probability for most frequent human HLAs. Conclusions This work provides a framework able to track down SARS-CoV-2 genomic variability.

scholarly journals A Novel Computational Framework to Predict the Impact of a Point Mutation on PDZ Domain Classification

2018 ◽  
Author(s):  
Muhammad Moinuddin ◽  
Wasim Aftab ◽  
Adnan Memic
Keyword(s):  
Usher Syndrome ◽  
Pdz Domain ◽  
Point Mutations ◽  
Similarity Measures ◽  
Bigram Frequency ◽  
Computational Techniques ◽  
Pdz Domains ◽  
Computational Framework ◽  
Class 1 ◽  
The Impact

AbstractPDZ domains represent one of the most common protein homology regions playing key roles in several diseases. Point mutations (PM) in amino acid primary sequence of PDZ domains can alter domain functions by affecting for example, downstream phosphorylation, a pivotal process in biology. Our goal in this present study was to introduce a novel approach to investigate how point mutations within the Class 1, Class 2 and Class 1–2 PDZ domains could affect the changes in binding with their partner ligands and hence affect their classification. We focused on features in PDZ domains of various species including human, rat and mouse. However, our work represents a generic computational framework that could be used to analyze PM in any given PDZ sequence. We have adopted two different approaches to investigate the impact of PM. In the first approach, we have developed a statistical model using bigram frequencies of amino acids and employed six different similarity measures to contrast the bigram frequency distribution of a wild type sequence relevant to its point mutants. In the next approach, we developed a statistical method that incorporates the impact of bigram frequency history associated with each mutational site that we call history weighted conditional change in probabilities. In this PM study, we observed that the history weighted method performs best when compared to all other methods studied in terms of picking up sites in PDZ domain where a PM could flip the class. We anticipate that this method will present a step forward towards computational techniques unveiling PDZ domain point mutants that largely affect the protein-ligand binding, specificity and affinity. We hope that this and future studies could aid therapy in which PDZ mutations have been implicated as the main disease drivers such as the Usher syndrome.

scholarly journals The Conformational Plasticity Vista of PDZ Domains

Life ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 123 ◽  
Author(s):  
Javier Murciano-Calles
Keyword(s):  
Pdz Domain ◽  
Adaptor Proteins ◽  
Pdz Domains ◽  
Post Translational Modifications ◽  
Multiple Partners ◽  
Conformational Plasticity ◽  
Modular Domain ◽  
Intrinsic Plasticity ◽  
Family Based ◽  
Living Organisms

The PDZ domain (PSD95-Discs large-ZO1) is a widespread modular domain present in the living organisms. A prevalent function in the PDZ family is to serve as scaffolding and adaptor proteins connecting multiple partners in signaling pathways. An explanation of the flexible functionality in this domain family, based just on a static perspective of the structure–activity relationship, might fall short. More dynamic and conformational aspects in the protein fold can be the reasons for such functionality. Folding studies indeed showed an ample and malleable folding landscape for PDZ domains where multiple intermediate states were experimentally detected. Allosteric phenomena that resemble energetic coupling between residues have also been found in PDZ domains. Additionally, several PDZ domains are modulated by post-translational modifications, which introduce conformational switches that affect binding. Altogether, the ability to connect diverse partners might arise from the intrinsic plasticity of the PDZ fold.

scholarly journals NHERF Links the N-Cadherin/Catenin Complex to the Platelet-derived Growth Factor Receptor to Modulate the Actin Cytoskeleton and Regulate Cell Motility

2007 ◽  
Vol 18 (4) ◽  
pp. 1220-1232 ◽  
Author(s):  
Christopher S. Theisen ◽  
James K. Wahl ◽  
Keith R. Johnson ◽  
Margaret J. Wheelock
Keyword(s):  
Cell Migration ◽  
Growth Factor ◽  
Kinase Inhibitor ◽  
Pdz Domain ◽  
Growth Factor Receptor ◽  
Cell Types ◽  
Platelet Derived Growth Factor ◽  
Binding Motif ◽  
Pdz Domains ◽  
Dependent Cell

Using phage display, we identified Na+/H+ exchanger regulatory factor (NHERF)-2 as a novel binding partner for the cadherin-associated protein, β-catenin. We showed that the second of two PSD-95/Dlg/ZO-1 (PDZ) domains of NHERF interacts with a PDZ-binding motif at the very carboxy terminus of β-catenin. N-cadherin expression has been shown to induce motility in a number of cell types. The first PDZ domain of NHERF is known to bind platelet-derived growth factor-receptor β (PDGF-Rβ), and the interaction of PDGF-Rβ with NHERF leads to enhanced cell spreading and motility. Here we show that β-catenin and N-cadherin are in a complex with NHERF and PDGF-Rβ at membrane ruffles in the highly invasive fibrosarcoma cell line HT1080. Using a stable short hairpin RNA system, we showed that HT1080 cells knocked down for either N-cadherin or NHERF had impaired ability to migrate into the wounded area in a scratch assay, similar to cells treated with a PDGF-R kinase inhibitor. Cells expressing a mutant NHERF that is unable to associate with β-catenin had increased stress fibers, reduced lamellipodia, and impaired cell migration. Using HeLa cells, which express little to no PDGF-R, we introduced PDGF-Rβ and showed that it coimmunoprecipitates with N-cadherin and that PDGF-dependent cell migration was reduced in these cells when we knocked-down expression of N-cadherin or NHERF. These studies implicate N-cadherin and β-catenin in cell migration via PDGF-R–mediated signaling through the scaffolding molecule NHERF.

scholarly journals Structural analysis of phosphorylation-associated interactions of MCC to Scribble PDZ domains

2018 ◽  
Author(s):  
Sofia Caria ◽  
Bryce Z Stewart ◽  
Patrick O Humbert ◽  
Marc Kvansakul
Keyword(s):  
Cell Polarity ◽  
Posttranslational Modifications ◽  
Adaptor Protein ◽  
Structural Basis ◽  
Binding Motif ◽  
Pdz Domains ◽  
Wild Type ◽  
Ligand Interactions ◽  
Direct Cell ◽  
The Impact

Scribble is a crucial adaptor protein that plays a pivotal role during establishment and control of cell polarity, impacting many physiological processes ranging from cell migration to immunity and organization of tissue architecture. Scribble harbors a leucine-rich repeat domain and four PDZ domains, which mediate most of Scribbles interactions with other proteins. It has become increasingly clear that posttranslational modifications substantially impact Scribble-ligand interactions, with phosphorylation being a major modulator of binding to Scribble. To better understand how Scribble PDZ domains direct cell polarity signalling and how phosphorylation impacts this process, we investigated Scribble interactions with MCC (mutated in colorectal cancer). We systematically evaluated the ability of all four individual Scribble PDZ domains to bind the PDZ-binding motif (PBM) of wild-type MCC as well as MCC phosphorylated at the -1 Ser position. We show that Scribble PDZ1 and PDZ3 are the major interactors with MCC, and that modifications to Ser at the -1 position in the MCC PBM only has a modest effect on binding to Scribble PDZ domains. We then examined the structural basis for these observations by determining the crystal structures of Scribble PDZ1 domain bound to both the wild-type MCC PBM as well as phosphorylated MCC. Our structures indicated that phospho-Ser at the -1 position in MCC is not involved in major contacts with Scribble PDZ1, and in conjunction with our affinity measurements suggest that the impact of phosphorylation at the -1 position of MCC extends beyond a simple modulation of the affinity for Scribble PDZ domains.

scholarly journals NHERF1/EBP50 Head-to-Tail Intramolecular Interaction Masks Association with PDZ Domain Ligands

2007 ◽  
Vol 27 (7) ◽  
pp. 2527-2537 ◽  
Author(s):  
Fabiana C. Morales ◽  
Yoko Takahashi ◽  
Safan Momin ◽  
Henry Adams ◽  
Xiaomin Chen ◽  
...  
Keyword(s):  
Intramolecular Interaction ◽  
Pdz Domain ◽  
Epithelial Tissue ◽  
Binding Motif ◽  
Pdz Domains ◽  
Erm Proteins ◽  
Epithelial Cancers ◽  
Associated Proteins ◽  
Α Helix

ABSTRACT Loss of cell polarity is one of the initial alterations in the development of human epithelial cancers. Na+/H+ exchanger regulatory factor (NHERF) homologous adaptors 1 and 2 are membrane-associated proteins composed of two amino (N)-terminal PDZ domains and an ezrin-radixin-moesin (ERM)-binding (EB) carboxyl (C)-terminal region. We describe here an intramolecular conformation of NHERF1/EBP50 (ERM-binding phosphoprotein 50) in which the C-terminal EB region binds to the PDZ2 domain. This novel head-to-tail conformation masked the interaction of both PDZ domains with PDZ domain-specific ligands, such as PTEN and β-catenin. An EB region composite structure comprising an α-helix ending in a PDZ-binding motif imparted opposite effects to NHERF1 associations, mediating binding to ERM proteins and inhibiting binding of PDZ domain ligands. The PDZ domain inhibition was released by prior association of ezrin with the EB region, a condition that occurs in vivo and likely disrupts NHERF1 head-to-tail interaction. In contrast, NHERF2 did not present a regulatory mechanism for protein complex formation. Functionally, NHERF1 is required to organize complexes at the apical membranes of polarized epithelial cells. The regulation of NHERF1 interactions at the apical membrane thus appears to be a dynamic process that is important for maintaining epithelial-tissue integrity.

Plasma membrane Ca2+-ATPase (PMCA) translocates to filopodia during platelet activation

2004 ◽  
Vol 91 (02) ◽  
pp. 325-333 ◽  
Author(s):  
Sidney Whiteheart ◽  
William Dean
Keyword(s):  
Plasma Membrane ◽  
Platelet Activation ◽  
Essential Role ◽  
Immunofluorescence Microscopy ◽  
Pdz Domain ◽  
Binding Motif ◽  
Local Concentration ◽  
Pdz Domains ◽  
Clot Retraction ◽  
Block Association

SummaryThe plasma membrane Ca2+-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca2+ in platelets. Recently we demonstrated that PMCA is recruited to the cytoskeleton by interacting with PDZ domains. In the present study we determined the subcellular localization of PMCA using immunofluorescence microscopy. In resting platelets PMCA was distributed over the entire plasma membrane. Upon activation with thrombin, PMCA was found in filopodia adjacent to the actin cytoskeleton. PMCA translocation to filopodia was prevented by a peptide containing the last 10 residues of PMCA4b, the predominate isoform of PMCA in platelets, which contains a known PDZ domain-binding motif and was previously shown to block association of PMCA with the cytoskeleton. Incorporation of the PMCA C-terminal peptide did not affect the rate or extent of platelet aggregation, but significantly enhanced the rate of clot retraction. These results show that PMCA association with the cytoskeleton during platelet activation results in translocation of this Ca2+-pump to filopodia and that this association may affect later stages of platelet activation. The consequence of PMCA translocation to filopodia is likely a reduction in the local concentration of free Ca2+ in these structures resulting in regulation of the rate of clot retraction.

scholarly journals The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e98124 ◽  
Author(s):  
Javier Murciano-Calles ◽  
Marta Marin-Argany ◽  
Eva S. Cobos ◽  
Sandra Villegas ◽  
Jose C. Martinez
Keyword(s):  
Pdz Domain ◽  
Post Translational Modifications ◽  
Neuronal Protein ◽  
The Third ◽  
Domain Structures ◽  
The Impact

scholarly journals Structural basis of the human Scribble-Vangl2 association in health and disease

2020 ◽  
Author(s):  
Jing Yuan How ◽  
Rebecca K. Stephens ◽  
Krystle Y.B. Lim ◽  
Patrick O. Humbert ◽  
Marc Kvansakul
Keyword(s):  
Cell Polarity ◽  
Neural Tube ◽  
Neural Tube Defect ◽  
Peptide Mapping ◽  
Pdz Domain ◽  
Structural Basis ◽  
Binding Motif ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Pdz Domains

AbstractScribble is a critical cell polarity regulator that has been shown to work as either an oncogene or tumor suppressor in a context dependent manner, and also impacts cell migration, tissue architecture and immunity. Mutations in Scribble lead to neural tube defects in mice and humans, which has been attributed to a loss of interaction with the planar cell polarity regulator Vangl2. We show that the Scribble PDZ domains 1, 2 and 3 are able to interact with the C-terminal PDZ binding motif of Vangl2 and have now determined crystal structures of these Scribble PDZ domains bound to the Vangl2 peptide. Mapping of mammalian neural tube defect mutations reveal that mutations located distal to the canonical PDZ domain ligand binding groove can not only ablate binding to Vangl2 but also disrupt binding to multiple other signaling regulators. Our findings suggest that PDZ-associated neural tube defect mutations in Scribble may not simply act in a Vangl2 dependent manner but as broad-spectrum loss of function mutants by disrupting the global Scribble-mediated interaction network.

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