Monomer/dimer transition of the caspase-recruitment domain of human Nod1.
ABSTRACT Nod1 is an essential cytoplasmic sensor for bacterial peptidoglycans in the innate immune system. The caspase-recruitment domain of Nod1 (Nod1_CARD) is indispensable for recruiting a downstream kinase, receptor-interacting protein 2 (RIP2), that activates nuclear factor-kappaB (NF-kappaB). The crystal structure of human Nod1_CARD at 1.9 A resolution reveals a novel homodimeric conformation. Our structural and biochemical analysis shows that the homodimerization of Nod1_CARD is achieved by swapping the H6 helices at the carboxy termini and stabilized by forming an interchain disulfide bond between the Cys39 residues of the two monomers in solution and in the crystal. In addition, we present experimental evidence for a pH-sensitive conformational change of Nod1_CARD. Our results suggest that the pH-sensitive monomer/dimer transition is a unique molecular property of Nod1_CARD.
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ABSTRACT: The caspase recruitment domain (CARD) is present in death-domain superfamily proteins involved in inflammation and apoptosis. BinCARD is named for its ability to interact with Bcl10 and inhibit downstream signalling. Human BinCARD is expressed as two isoforms that encode the same N-terminal CARD region but which differ considerably in their C-termini. Both isoforms are expressed in immune cells, although BinCARD-2 is much more highly expressed. Crystals of the CARD fold common to both had low symmetry (space group P1). Molecular replacement was unsuccessful in this low-symmetry space group and, as the construct contains no methionines, first one and then two residues were engineered to methionine for MAD phasing. The double-methionine variant was produced as a selenomethionine derivative, which was crystallized and the structure was solved using data measured at two wavelengths. The crystal structures of the native and selenomethionine double mutant were refined to high resolution (1.58 and 1.40 Å resolution, respectively), revealing the presence of a cis-peptide bond between Tyr39 and Pro40. Unexpectedly, the native crystal structure revealed that all three cysteines were oxidized. The mitochondrial localization of BinCARD-2 and the susceptibility of its CARD region to redox modification points to the intriguing possibility of a redox-regulatory role.Acta Crystallographica Section D Biological Crystallography 05/2013; 69(Pt 5):774-84. · 12.67 Impact Factor
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ABSTRACT: Following activation, the cytoplasmic pattern recognition receptor NOD1 interacts with its adaptor protein RIP2 to propagate immune signalling and initiate a pro-inflammatory immune response. This interaction is mediated by the caspase recruitment domain (CARD) of both proteins. Polymorphisms in immune proteins can affect receptor function and predispose individuals to specific autoinflammatory disorders. In this report, we have shown that mutations in helix 2 of the CARD of NOD1 disrupt receptor function, but do not interfere with RIP2 interaction. In particular Asn43Ser, a rare polymorphism, results in receptor dysfunction despite retaining normal cellular localisation, protein folding, and an ability to interact with RIP2. Mutation of Asn43 results in an increased tendency to form dimers, which we propose is the source of this dysfunction. We also demonstrate that mutation of Lys443 and Tyr474 in RIP2 disrupts the interaction with NOD1. Mapping the key residues involved in the interaction between NOD1 and RIP2 to the known structures of CARD complexes reveals the likely involvement of both type I and type III interfaces in the NOD1:RIP2 complex. Overall we demonstrate that the NOD1:RIP2 signalling axis is more complex than previously assumed, that simple engagement of RIP2 is insufficient to mediate signalling, and that the interaction between NOD1 and RIP2 constitutes multiple CARD:CARD interfaces.The Journal of biological chemistry. 06/2014;
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ABSTRACT: Activation and signal transduction in the Nucleotide binding, leucine-rich repeat containing receptor (NLR) family needs to be tightly regulated in order to control the inflammatory response to exogenous and endogenous danger signals. Phosphorylation is a common cellular mechanism of regulation that has recently been shown to be important in signalling in another family of cytoplasmic pattern recognition receptors, the RIG-I like receptors. In addition, single nucleotide polymorphisms can alter receptor activity, potentially leading to dysfunction and/or a predisposition to inflammatory barrier diseases. We have computationally analysed the N-terminus of NOD1 and found seven theoretical phosphorylation sites in, or immediately before, the NOD1 Caspase Activation Domain (CARD). Two of these, serine 7 and tyrosine 49 are also found as rare polymorphisms in the African-American population and European-American populations respectively. Mutating serine 7 to either an aspartic acid or an asparagine to mimic the potential impact of phosphorylation or the polymorphism respectively did not affect the response of NOD1 to ligand-mediated NFkappaB signalling. The NOD1 polymorphism S7N does not interfere with receptor function in response to ligand stimulation.BMC Research Notes 03/2014; 7(1):124.