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) from the innate immune receptor NOD1 was crystallized with Ubiquitin (Ub). NOD1 CARD was present as a helix-swapped homodimer similar to other structures of NOD1 CARD, and Ub monomers formed a homodimer similar in conformation to Lys48-linked di-Ub. The interaction between NOD1 CARD and Ub in the crystal was mediated by novel binding sites on each molecule. Comparisons of these sites to previously identified interaction surfaces on both molecules were made along with discussion of their potential functional significance.PLoS ONE 01/2014; 9(8):e104017. · 3.53 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: The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed.Acta Crystallographica Section D Biological Crystallography 07/2014; 70(Pt 7):2007-18. · 12.67 Impact Factor