Structural models of DYNLL1 with interacting partners: African swine fever virus protein p54 and postsynaptic scaffolding protein gephyrin

García-Mayoral, Mâria Flor; Rodrı́guez-Crespo, Ignacio; Bruix, Marta

doi:10.1016/j.febslet.2010.11.027

Cited by 20 publications

(19 citation statements)

References 15 publications

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“…NMR Spectroscopy-Recombinant protein was expressed in bacteria and labeled with 15 NH 4 Cl as reported previously (35,36). Solutions of the PDZ domain with final concentrations in the range of 100 -200 M were prepared in water with 10% D 2 O in buffer 100 mM KH 2 PO 4 , pH 6.5.…”

Section: Reagents-mentioning

confidence: 99%

Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase

Merino-Gracia

Costas-Insua

Canales

et al. 2016

Journal of Biological Chemistry

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Neuronal nitric-oxide synthase, unlike its endothelial and inducible counterparts, displays a PDZ (PSD-95/Dlg/ZO-1) domain located at its N terminus involved in subcellular targeting. The C termini of various cellular proteins insert within the binding groove of this PDZ domain and determine the subcellular distribution of neuronal NOS (nNOS). The molecular mechanisms underlying these interactions are poorly understood because the PDZ domain of nNOS can apparently exhibit class I, class II, and class III binding specificity. In addition, it has been recently suggested that the PDZ domain of nNOS binds with very low affinity to the C termini of target proteins, and a necessary simultaneous lateral interaction must take place for binding to occur. We describe herein that the PDZ domain of nNOS can behave as a bona fide class III PDZ domain and bind to C-terminal sequences with acidic residues at the P ؊2 position with low micromolar binding constants. Binding to C-terminal sequences with a hydrophobic residue at the P ؊2 position plus an acidic residue at the P ؊3 position (class II) can also occur, although interactions involving residues extending up to the P ؊7 position mediate this type of binding. This promiscuous behavior also extends to its association to class I sequences, which must display a Glu residue at P ؊3 and a Thr residue at P ؊2 . By means of site-directed mutagenesis and NMR spectroscopy, we have been able to identify the residues involved in each specific type of binding and rationalize the mechanisms used to recognize binding partners. Finally, we have analyzed the high affinity association of the PDZ domain of nNOS to claudin-3 and claudin-14, two tight junction tetraspan membrane proteins that are essential components of the paracellular barrier. Neuronal NOS (nNOS)2 is expressed constitutively in specific neurons of the brain and in the spinal cord, peripheral nitrergic nerves, epithelial cells of various organs, pancreatic islet cells, and vascular smooth muscle (1). nNOS differs from the two other mammalian isoforms, endothelial NOS and inducible NOS by an additional ϳ300-residue N-terminal extension mostly involved in specific subcellular targeting. The N terminus of nNOS contains a PDZ domain (first found in the proteins PSD-95, Dlg, and ZO-1) (2), a ␤-hairpin module that associates to ␣1-syntrophin and PSD-95 (3), a DYNLL1 binding site (4), and a stretch known to bind to repeats R16/R17 of dystrophin (5). Brain nNOS is found in particulate and soluble forms in cells, and the differential subcellular localization of nNOS in various tissues may contribute to its diverse functions. Reinforcement of the idea that the subcellular targeting is exquisitely governed by this N-terminal extension came from the observation that an N-terminal deletion mutant of nNOS is an active, mislocalized enzyme (6). In the nervous system, the PDZ domain of PSD-95 is known to couple NMDA receptors to nNOS so that ⅐ NO release becomes reversibly regulated by Ca 2ϩ /calmodulin binding (1). PDZ domains are modula...

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Section: Reagents-mentioning

confidence: 99%

Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase

Merino-Gracia

Costas-Insua

Canales

et al. 2016

Journal of Biological Chemistry

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“…LC8 interacts with a variety of viral proteins to facilitate movement of viral components (19,20,(28)(29)(30)(31). The rabies virus P protein, which serves as a rabies virus polymerase cofactor, also interacts with LC8 (32,33).…”

mentioning

confidence: 99%

Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

Luthra

Jordan

Leung

et al. 2015

J Virol

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bEbola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8. E bola virus (EBOV) VP35 is a multifunctional protein critical for both viral innate immune evasion and viral RNA synthesis (1, 2). It contains an N-terminal oligomerization domain and a C-terminal double-stranded RNA (dsRNA) binding domain referred to as the interferon inhibitory domain (IID) (3, 4). VP35 acts as an interferon antagonist, using both dsRNA-binding-dependent and -independent mechanisms, to block the RIG-I-like receptor signaling pathways (5-9). VP35 also participates with the viral large protein (L), the enzymatic component of the RNA-dependent RNA polymerase (RDRP) complex, VP30, and nucleoprotein (NP) in viral RNA synthesis (10, 11). VP35 is critical in this role because it mediates the interaction between L and the RNA template-associated NP (2,(12)(13)(14).VP35 can interact with the highly conserved 8-kDa cytoplasmic dynein light-chain (LC8) protein (15). LC8 is a subunit of the cytoplasmic dynein motor complex, which plays an important role in the microtubule-associated intracellular retrograde transport system (16,17). It also can exist in a soluble form without association with the dynein motor or microtubule (18). LC8 exists as a dimer containing two identical grooves located at opposite faces of the protein dimer interface (19). These grooves bind to proteins containing consensus motifs of either (K/S)XTQT or G (I/V)QVD (20). VP35 contains an SQTQT motif that is required for LC8 interaction (15). However, LC8 binding was not required for, nor did it inhibit, VP35 interferon (IFN) antagonist function (15,21). Therefore, the significance of the VP35-LC8 interaction has remained undefined.Here we characterized the interaction between LC8 and EBOV VP35 and provide evidence that the interaction stabilizes the VP35 N-terminal oligomerization domain to enhance viral RNA synthesis. The VP35-LC8 interaction was first validated by coimmunoprecipitation (co-IP). Briefly, HEK293T cells were transfected with plasmids expressing hemagglutinin (HA)-tagged LC8 (500 ng) and either full-length FLAG-tagged VP35 (1 g) (GenBank accession no. AF086833.2) or a VP35 truncation mutant (1 g) corresponding to the VP35 N-terminal domain (FLAG-VP35-N, residues 1 to 218) or the IID (FLAG-VP35-C, residues 219 to 340). Twenty-four hours posttransfection, anti-FLAG immunoprecipitations were performed. Both wild-type (WT) VP35 and VP35-N coprecipitated with LC8, confirming that VP35 binds LC8 via its N terminus (Fig. 1A). Mutation to alanine of T73 or Q74 within the VP35 71-SQTQT-75 motif disrupted or...

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“…Next, to validate the biological significance of putative Pilin-DYNLL1 binding motif, 3D structural comparisons of previously reported DYNLL1 binding motifs for BimEL, Adenain, DNMT3A, EML3, nNOS, PAK1, Rack1, Vaccinia polymerase P protein of Mokola and P protein Rabies viruses [2], [6], [7] were performed. These structures were superimposed for comparing the binding motif conservation structurally to further narrow down the interaction crosstalk.…”

Section: Resultsmentioning

confidence: 99%

Comparative Molecular Docking Analysis of Cytoplasmic Dynein Light Chain DYNLL1 with Pilin to Explore the Molecular Mechanism of Pathogenesis Caused by Pseudomonas aeruginosa PAO

et al. 2013

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Cytoplasmic dynein light chain 1 (DYNLL1) is a component of large protein complex, which is implicated in cargo transport processes, and is known to interact with many cellular and viral proteins through its short consensus motif (K/R)XTQT. Still, it remains to be explored that bacterial proteins also exhibit similar recognition sequences to make them vulnerable to host defense mechanism. We employed multiple docking protocols including AUTODOCK, PatchDock, ZDOCK, DOCK/PIERR and CLUSPRO to explore the DYNLL1 and Pilin interaction followed by molecular dynamics simulation assays. Subsequent structural comparison of the predicted binding site for DYNLL1-Pilin complex against the experimentally verified DYNLL1 binding partners was performed to cross check the residual contributions and to determine the binding mode. On the basis of in silico analysis, here we describe a novel interaction of DYNLL1 and receptor binding domain of Pilin (the main protein constituent of bacterial type IV Pili) of gram negative bacteria Pseudomonas aeruginosa (PAO), which is the third most common nosocomial pathogen associated with the life-threatening infections. Evidently, our results underscore that Pilin specific motif (KSTQD) exhibits a close structural similarity to that of Vaccinia virus polymerase, P protein Rabies and P protein Mokola viruses. We speculate that binding of DYNLL1 to Pilin may trigger an uncontrolled inflammatory response of the host immune system during P. aeruginosa chronic infections thereby opening a new pioneering area to investigate the role of DYNLL1 in gram negative bacterial infections other than viral infections. Moreover, by manifesting a strict correspondence between sequence and function, our study anticipates a novel drug target site to control the complications caused by P. aeruginosa infections.

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Structural models of DYNLL1 with interacting partners: African swine fever virus protein p54 and postsynaptic scaffolding protein gephyrin

Cited by 20 publications

References 15 publications

Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase

Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase

Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

Comparative Molecular Docking Analysis of Cytoplasmic Dynein Light Chain DYNLL1 with Pilin to Explore the Molecular Mechanism of Pathogenesis Caused by Pseudomonas aeruginosa PAO

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