PLoS One. 2012;7(3):e34375. Epub 2012 Mar 28.

The tandem CARDs of NOD2: intramolecular interactions and recognition of RIP2.External

Fridh, V., Rittinger, K.,
--- - Division of Molecular Structure, MRC-National Institute for Medical Research, London, United Kingdom.
Caspase recruitment domains (CARDs) are homotypic protein interaction modules that link the stimulus-dependent assembly of large signaling platforms such as inflammasomes to the activation of downstream effectors that often include caspases and kinases and thereby play an important role in the regulation of inflammatory and apoptotic signaling pathways. NOD2 belongs to the NOD-like (NLR) family of intracellular pattern recognition receptors (PRR) and induces activation of the NF-kappaB pathway in response to the recognition of bacterial components. This process requires the specific recognition of the CARD of the protein kinase RIP2 by the tandem CARDs of NOD2. Here we demonstrate that the tandem CARDs of NOD2 are engaged in an intramolecular interaction that is important for the structural stability of this region. Using a combination of ITC and pull-down experiments we identify distinct surface areas that are involved in the intramolecular tandem CARD interaction and the interaction with the downstream effector RIP2. Our findings indicate that while CARDa of NOD2 might be the primary binding partner of RIP2 the two CARDs of NOD2 do not act independently of one another but may cooperate to from a binding surface that is distinct from that of single CARDs.
PMID: 22470564External
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
In vitro interaction
  DD1 DD2 Reference
Family DD1 DD2 Method Species Region Expression Species Region Expression
CARD NOD1 Link RIPK2 GST fusion protein pull-down Not specified 17-138 E.coli Not specified 435-528 E.coli 22470564
CARD NOD2 Link NOD2 In vitro purification protein assembly(Isothermal titration calorimety) Not specified 28-119 E.coli Not specified 120-218 E.coli 22470564
CARD NOD2 Link RIPK2 GST fusion protein pull-down Not specified 28-218 E.coli Not specified 435-528 E.coli 22470564
(Link: click this icon to show interactions only between the two corresponding DDs)
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
Characterization
  Stoichiometry Affinity Protein-Protein interface Reference
Family DD1 DD2 Method Mutation Complex structure
CARD NOD1 Link RIPK2 GST fusion protein pull-down D461A, E472A, E475A, E492A (RIPK2) 22470564
CARD NOD2 Link NOD2 Isothermal titration calorimety 1:1 1μM 22470564
CARD NOD2 Link RIPK2 GST fusion protein pull-down R38A, R86A (NOD2) D461A, E472A, D473A, E475A, D492A (RIPK2) 22470564
(Link: click this icon to show interactions only between the two corresponding DDs)