NOD1CARD Might Be Using Multiple Interfaces for RIP2-Mediated CARD-CARD Interaction: Insights from Molecular Dynamics Simulation

Maharana, Jitendra; Pradhan, Sukanta Kumar; De, Sachinandan

doi:10.1371/journal.pone.0170232

Cited by 18 publications

(34 citation statements)

References 36 publications

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“…The interaction of NOD1 and NOD2 with RIPK2 is mediated by heterotypic CARD:CARD interactions, involving residues in the exposed surfaces of the CARD domains of RIPK2 and NOD1/2 (Maharana, Pradhan et al, 2017, Manon, Favier et al, 2007, Mayle, Boyle et al, 2014. In vitro, this can result in stable rope-like structures, which were recently proposed to be platforms for subsequent NF-κB activation (Gong, Long et al, 2018, Pellegrini, Desfosses et al, 2018.…”

Section: Introductionmentioning

confidence: 99%

XIAP controls RIPK2 signaling by preventing its deposition in speck-like structures

Ellwanger

Arnold

Briese

et al. 2019

Preprint

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The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for linking activation of the pattern recognition receptors NOD1 and NOD2 to cellular signaling events.Recently, it was shown that RIPK2 forms higher order molecular structures in vitro, which were proposed to activate signaling. Here, we demonstrate that RIPK2 forms detergent insoluble complexes in the cytosol of host cells upon infection with invasive enteropathogenic bacteria. Formation of these structures occurred after NF-κB activation and depends on the CARD of NOD1 or NOD2. Complex formation upon activation was dependent on RIPK2 autophosphorylation at Y474 and influenced by phosphorylation at S176. Inhibition of activity of the cIAP protein XIAP induced spontaneous complex formation of RIPK2 but blocked NOD1-dependet NF-κB activation. Using immunoprecipitation, we identified 14-3-3 proteins as novel binding partners of non-activated RIPK2, whereas complexed RIPK2 was bound by the prohibitin proteins Erlin-1 and Erlin-2.Taken together, our work reveals novel roles of XIAP, 14-3-3 and Erlin proteins in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling. AUTHOR CONTRIBUTIONSCA, KE, SB and IK performed the experiments.JP performed MS analysis and data processing.TAK conceived and supervised the study.CA, KE and TAK wrote and edited the manuscript. CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.A: Indirect immunofluorescence micrographs of HeLa EGFP-RIPK2 cells infected for 2 h with S. flexneri M90T. Staining for XIAP, EGFP-RIPK2, and merge with DNA-staining is shown.Co-localization is shown at higher magnification in the inlay. Scale bar = 10 µm. B: Fluorescence micrographs of HeLa EGFP-RIPK2 cells, treated for 48 h with a XIAP siRNA or a non-targeting siRNA, following infection with S. flexneri M90T for 2 h or left uninfected. Cells were treated with 1 µg/ml doxycycline for 24 h prior infection. EGFP-RIPK2 and merge with DNA-staining is shown. Scale bar = 10 µm. C: IL-8 release in the supernatants from HeLa EGFP-RIPK2 cells, treated for 48 h with a XIAP siRNA or a non-targeting siRNA, following infection with S. flexneri M90T for 6 h. Mean of n=2 with S.D. is shown. D: Immunoblot analysis of HeLa EGFP-RIPK2 cells infected with S. flexneri M90T or BS176 for the indicated time. Immunoblot was probed with anti-RIPK2 antibody, anti-XIAP, and anti-β-tubulin as loading control. E: Left panel: Fluorescence micrographs of HeLa EGFP-RIPK2 cells, transfected with 500 ng Ubi-SMAC-fl-pDS-RED, Ubi-SMAC-tr-pDS-RED, or Ubi-SMAC-AVPI-pDS-RED plasmid and treated with 1 µg/ml doxycycline for 24 h. EGFP-RIPK2, SMAC-dsRed, and merge with DNA-staining is shown. Scale bar = 10 µm. Right panel: Immunoblot analysis of cells prepared as in the left panel. Protein levels were detected using an anti-RIPK2 antibody and anti-β-tubulin as loading control.Figure 6: Phosphorylation of RIPK2 at S176 and Y474 contribute to RIPosome formation. A: Fluorescence microg...

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Section: Introductionmentioning

confidence: 99%

XIAP controls RIPK2 signaling by preventing its deposition in speck-like structures

Ellwanger

Arnold

Briese

et al. 2019

Preprint

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“…A sequence alignment of ASC PYD , NLRP3 PYD , and POP1 (using MAFFT) shows the conserved charged residues of both the surfaces (Figure D). Molecular docking of NLRP3 PYD and POP1 has been performed using PyMOL superimposition protocol . A total of two different heterodimeric complexes of POP1‐NLRP3 PYD were created.…”

Section: Resultsmentioning

confidence: 99%

“…Molecular docking of NLRP3 PYD and POP1 has been performed using PyMOL superimposition protocol. 34 Figure S1A). To investigate the involvement of type-II and type-III interfaces, we also modeled four heterotrimeric complexes ( Figure S1B) considering ASC PYD filament structure Figure 3B).…”

Section: Identification Of Protein-binding Surface and Molecular Domentioning

confidence: 99%

POP1 might be recruiting its type‐Ia interface for NLRP3‐mediated PYD‐PYD interaction: Insights from MD simulation

Maharana

Vats

Gautam

et al. 2017

J of Molecular Recognition

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Inflammasomes are multiprotein caspase-activating complexes that enhance the maturation and release of proinflammatory cytokines (IL-1β and IL-18) in response to the invading pathogen and/or host-derived cellular stress. These are assembled by the sensory proteins (viz NLRC4, NLRP1, NLRP3, and AIM-2), adaptor protein (ASC), and effector molecule procaspase-1. In NLRP3-mediated inflammasome activation, ASC acts as a mediator between NLRP3 and procaspase-1 for the transmission of signals. A series of homotypic protein-protein interactions (NLRP3 :ASC and ASC :CASP1 ) propagates the downstream signaling for the production of proinflammatory cytokines. Pyrin-only protein 1 (POP1) is known to act as the regulator of inflammasome. It modulates the ASC-mediated inflammasome assembly by interacting with pyrin domain (PYD) of ASC. However, despite similar electrostatic surface potential, the interaction of POP1 with NLRP3 is obscured till date. Herein, to explore the possible PYD-PYD interactions between NLRP3 and POP1, a combined approach of protein-protein docking and molecular dynamics simulation was adapted. The current study revealed that POP1's type-Ia interface and type-Ib interface of NLRP3 might be crucial for 1:1 PYD-PYD interaction. In addition to type-I mode of interaction, we also observed type-II and type-III interaction modes in two different dynamically stable heterotrimeric complexes (POP1-NLRP3-NLRP3 and POP1-NLRP3-POP1). The inter-residual/atomic distance calculation exposed several critical residues that possibly govern the said interaction, which need further investigation. Overall, the findings of this study will shed new light on hitherto concealed molecular mechanisms underlying NLRP3-mediated inflammasome, which will have strong future therapeutic implications.

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“…The main function of CARDs is to take part in the CARD-CARD or CARD-DD interactions. These interactions are known to have the ionic nature: surfaces of most CARDs reveal the expressed asymmetry of electrostatic properties—extended positively and negatively charged regions are observed (b-face and a-face) [ 34 ]. In the case of RIP2CARD the b-face is formed by R443 and R487—residue which is also conserved in most CARD domains.…”

Section: Discussionmentioning

confidence: 99%

CARD domain of rat RIP2 kinase: Refolding, solution structure, pH-dependent behavior and protein-protein interactions

et al. 2018

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RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune response against gram-negative and gram-positive bacteria via its caspase recruitment domain (CARD). This makes RIP2 a prospective target for novel therapies, aimed to modulate the inflammatory diseases and neurogenesis/neurodegeneration. Several studies report the problems with the stability of human RIP2 CARD and its production in bacterial hosts, which is a prerequisite for the structural investigation with solution NMR spectroscopy. In the present work, we report the high yield production and refolding protocols and resolve the structure of rat RIP2 CARD. The structure reveals the important differences to the previously published conformation of the homologous human protein. Using solution NMR, we characterized the intramolecular mobility and pH-dependent behavior of RIP2 CARD, and found the propensity of the protein to form high-order oligomers at physiological pH while being monomeric under acidic conditions. The oligomerization of protein may be explained, based on the electrostatic properties of its surface. Analysis of the structure and sequences of homologous proteins reveals the residues which are significant for the unusual fold of RIP2 CARD domains from different species. The high-throughput protein production/refolding protocols and proposed explanation for the protein oligomerization, provide an opportunity to design the stabilized variants of RIP2 CARD, which could be used to study the structural details of RIP2/NOD1/NOD2 interaction and perform the rational drug design.

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NOD1CARD Might Be Using Multiple Interfaces for RIP2-Mediated CARD-CARD Interaction: Insights from Molecular Dynamics Simulation

Cited by 18 publications

References 36 publications

XIAP controls RIPK2 signaling by preventing its deposition in speck-like structures

XIAP controls RIPK2 signaling by preventing its deposition in speck-like structures

POP1 might be recruiting its type‐Ia interface for NLRP3‐mediated PYD‐PYD interaction: Insights from MD simulation

CARD domain of rat RIP2 kinase: Refolding, solution structure, pH-dependent behavior and protein-protein interactions

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