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Structural and thermodynamic origins of distinct ligand specificity of two homologous PDZ domainsShepherd, Tyson Robert 01 July 2011 (has links)
Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a previously uncharacterized post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Here we report on the structure, specificity, and function of the Tiam1 and Tiam2 PDZ domains.
A consensus PDZ-binding motif for Tiam1 was used to predict that two cell adhesion proteins, Syndecan 1 (Sdc1) and Caspr4, are potential Tiam1 PDZ domain binding proteins. Binding interactions were confirmed using fluorescence- and NMR- based binding experiments. The Tiam1 PDZ domain in complex with the C-terminal tails of Sdc1 and phosphorylated Sdc1 were solved using X-ray crystallography. Results showed four residues in two binding pockets in the PDZ domain are important for specificity. Cell biological analysis confirmed the Tiam1/Sdc1 interaction and showed that the PDZ domain has a function in cell-matrix adhesion and cell migration.
The four residues deemed important determinants of Tiam1 PDZ domain specificity are not conserved in Tiam2. A combinatorial peptide screen, in combination with biophysical studies, identified a consensus binding sequence for both PDZ domains. Analysis of these consensus sequences and binding assays with peptides derived from native proteins indicated that these two PDZ domains have overlapping but distinct specificities - the Tiam2 PDZ domain was found to bind Caspr4 and neurexin1 but not Sdc1. Additionally, the Tiam2 PDZ domain exhibits significant flexibility in two different regions, a feature not seen in Tiam1.
Double-mutant cycle analysis of the four important residues revealed ligand- dependent energetic couplings. Mutating all four residues switched the ligand specificity to that of Tiam2. Analysis of Tiam-family PDZ domain sequences indicated that the PDZ domains segregate into four distinct families based on the residues studied here. A set of "evolved peptides" was used to show the PDZ domain interactions are cooperative throughout the binding pocket in a ligand-specific manner. Collectively, our data suggest that Tiam family proteins have highly evolved PDZ domain/ligand interfaces with distinct specificities and that they have disparate PDZ domain-dependent biological functions.
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Étude computationnelle du domaine PDZ de Tiam1 / Computational study of the Tiam1 PDZ domainPanel, Nicolas 07 November 2017 (has links)
Les interactions protéine-protéine sont souvent contrôlées par de petits domaines protéiques qui régulent les chemins de signalisation au sein des cellules eucaryotes. Les domaines PDZ sont parmi les domaines les plus répandus et les plus étudiés. Ils reconnaissent spécifiquement les 4 à 10 acides aminés C-terminaux de leurs partenaires. Tiam1 est un facteur d'échange de GTP de la protéine Rac1 qui contrôle la migration et la prolifération cellulaire et dont le domaine PDZ lie les protéines Syndecan-1 (Sdc1), Caspr4 et Neurexine. Des petits peptides ou des molécules peptidomimétiques peuvent potentiellement inhiber ou moduler son activité et être utilisés à des fins thérapeutiques. Nous avons appliqué des approches de dessin computationnel de protéine (CPD) et de calcul d'énergie libre par simulations dynamique moléculaire (DM) pour comprendre et modifier sa spécificité. Le CPD utilise un modèle structural et une fonction d'énergie pour explorer l'espace des séquences et des structures et identifier des variants protéiques ou peptidiques stables et fonctionnels. Nous avons utilisé le programme de CPD Proteus, développé au laboratoire, pour redessiner entièrement le domaine PDZ de Tiam1. Les séquences générées sont similaires à celles des domaines PDZ naturels, avec des scores de similarité et de reconnaissance de pli comparables au programme Rosetta, un outil de CPD très utilisé. Des séquences contenant environ 60 positions mutées sur 90, ont été testées par simulations de DM et des mesures biophysiques. Quatre des cinq séquences testées expérimentalement (par nos collaborateurs) montrent un dépliement réversible autour de 50°C. Proteus a également déterminer correctement la spécificité de la liaison de quelques variants protéiques et peptidiques. Pour étudier plus finement la spécificité, nous avons paramétré un modèle d'énergie libre semi-empirique de Poisson-Boltzmann ayant la forme d'une énergie linéaire d'interaction, ou PB/LIE, appliqué à des conformations issues de simulations de DM en solvant explicite de complexes PDZ:peptide. Avec trois paramètres ajustables, le modèle reproduit correctement les affinités expérimentales de 41 variants, avec une erreur moyenne absolue de 0,4~kcal/mol, et donne des prédictions pour 10 nouveaux variants. Le modèle PB/LIE a ensuite comparé à la méthode non-empirique de calcul d'énergie libre par simulations alchimiques, qui n'a pas de paramètre ajustable et qui prédit correctement l'affinité de 12 complexes Tiam1:peptide. Ces outils et les résultats obtenus devraient nous permettre d'identifier des peptides inhibiteurs et auront d'importantes retombées pour l'ingénierie des interactions PDZ:peptide. / Small protein domains often direct protein-protein interactions and regulate eukaryotic signalling pathways. PDZ domains are among the most widespread and best-studied. They specifically recognize the 4-10 C-terminal amino acids of target proteins. Tiam1 is a Rac GTP exchange factor that helps control cellmigration and proliferation and whose PDZ domain binds the proteins syndecan-1 (Sdc1), Caspr4, and Neurexin. Short peptides and peptidomimetics can potentially inhibit or modulate its action and act as bioreagents or therapeutics. We used computational protein design (CPD) and molecular dynamics (MD) free energy simulations to understand and engineer its peptide specificity. CPD uses a structural model and an energy function to explore the space of sequences and structures and identify stable and functional protein or peptide variants. We used our in-house Proteus CPD package to completely redesign the Tiam1 PDZ domain. The designed sequences were similar to natural PDZ domains, with similarity and fold recognition scores comarable to the widely-used Rosetta CPD package. Selected sequences, containing around 60 mutated positions out of 90, were tested by microsecond MD simulations and biophysical experiments. Four of five sequences tested experimentally (by our collaborators) displayed reversible unfolding around 50°C. Proteus also accurately scored the binding specificity of several protein and peptide variants. As a more refined model for specificity, we parameterized a semi-empirical free energy model of the Poisson-Boltzmann Linear Interaction Energy or PB/LIE form, which scores conformations extracted from explicit solvent MD simulations of PDZ:peptide complexes. With three adjustable parameters, the model accurately reproduced the experimental binding affinities of 41 variants, with a mean unsigned error of just 0.4 kcal/mol, andgave predictions for 10 new variants. The PB/LIE model was tested further by comparing to non-empirical, alchemical, MD free energy simulations, which have no adjustable parameters and were found to give chemical accuracy for 12 Tiam1:peptide complexes. The tools and insights obtained should help discover new tight binding peptides or peptidomimetics and have broad implications for engineering PDZ:peptide interactions.
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Identification and validation of small molecule inhibitors for the Tiam1/SDC1 interactionLopez, Josue Alan 01 May 2014 (has links)
No description available.
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Targeting Pleckstrin Homology Domains for the Inhibition of Cancer Growth and MetastasisMoses, Sylvestor Andrea January 2013 (has links)
Pleckstrin homology (PH) domains are structurally conserved domains, which generally bind to phosphatidylinositol phosphate (PtdInsP) lipids. They are present in a variety of proteins, including those that are upregulated in cancer growth and metastasis, and represent a crucial component of intracellular signaling cascades and membrane translocation. Thus, they may be considered as attractive targets for cancer drug therapy. AKT (protein kinase B), a pleckstrin homology lipid binding domain and a serine/threonine kinase-containing protein, is a key component of the phophatidylinositol-3-kinase (PI3K)/AKT cell survival signaling pathway which is activated in a variety of cancers, including prostate, pancreatic, and skin cancers. In this study, I report the finding of a novel inhibitor of AKT; PH-427. I describe its effects on binding to the PH domain of AKT thus preventing its binding to PtdIns3-P at the plasma membrane and subsequent activation. In vivo testing of the drug led to reduction of tumor size and numbers in a mouse pancreatic cancer model. Additional testing of PH-427 on squamous cell carcinomas revealed that the drug is able to reduce tumor burden and multiplicity in vivo when topically applied. Thus, we demonstrate proof-of-principle in targeting PH domains as a viable cancer drug therapy option. The effects of PH-427 raised the intriguing possibility that targeting PH domains may have beneficial effects in other signaling pathways with PH domain-containing proteins. Guanine exchange factors (GEFs) contain a Dbl homology (DH) domain and a PH domain and have been shown to be involved in the process of metastasis. More specifically, RacGEFs activate Rac1 GTPase by facilitating the exchange of GDP to GTP. Over-expression of certain GEFs has been shown to contribute to increased malignancy in a variety of cancers. T-lymphoma invasion and metastasis-inducing protein-1 (Tiam1) is a highly conserved GEF and contains an N-terminal pleckstrin homology domain (nPH) and a DH/C-terminal PH domain (cPH). Tiam1 has been found to be over-expressed in several cancers, including breast, colon and prostate cancers. In this study, I describe the identification, development, experimental testing, and potential mechanism of action of novel small molecule inhibitors targeting the RacGEF Tiam1 to inhibit prostate cancer bone metastasis.
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Auto-inhibition mechanism of the guanine nucleotide exchange factor Tiam1Xu, Zhen 01 August 2016 (has links)
The Rho family of guanosine triphosphatases (GTPases) function as binary molecular switches, which play an important role in the regulation of actin cytoskeleton rearrangement and are involved in several critical cellular processes including cell adhesion, division and migration. Rho GTPases are specifically activated by their associated guanine nucleotide exchange factors (RhoGEFs). Dysregulation of RhoGEFs function through mutation or overexpression has been implicated in oncogenic transformation of cells and linked to several kinds of invasive and metastatic forms of cancer. T-cell lymphoma invasion and metastasis 1 (Tiam1) is a multi-domain Dbl family GEF protein and specifically activates Rho GTPase Rac1 through the catalytic Dbl homology and Pleckstrin homology (DH-PH) bi-domain. Previous works have shown that the nucleotide exchange function of the full-length Tiam1 is auto-inhibited and can be activated by N-terminal truncation, phosphorylation and protein-protein interactions. However, the molecular mechanisms of Tiam1 GEF auto-inhibition and activation have not yet been determined. In this study, the N-terminal PH-CC-Ex domain of Tiam1 is shown to directly inhibit the GEF function of the catalytic DH-PH domain in vitro. Using fluorescencebased kinetics experiments, we demonstrate that the auto-inhibition of Tiam1 GEF function occurs by a competitive inhibition model. In this model, the maximum velocity of catalytic activity remains unchanged, but the Michaelis-Menten constant of the auto-inhibited Tiam1 (the PH-PH fragment) on the substrate Rac1 is increased compared to the activated Tiam1 (the catalytic DH-PH domain alone). Through small angle X-ray scattering (SAXS), the structure of auto-inhibited Tiam1 (the PH-PH fragment) is shown to form a closed conformation in which the catalytic DH-PH domain is blocked by the N-terminal PH-CC-Ex domain. Taken together, these findings demonstrate the molecular mechanism of Tiam1 GEF autoinhibition in which the PH-CC-Ex domain of Tiam1 inhibits its GEF function by preventing the substrate Rho GTPase Rac1 from accessing the catalytic DH-PH bi-domain.
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