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: 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: E3 ligases mediate the covalent attachment of ubiquitin to target proteins thereby enabling ubiquitin-dependent signaling. Unraveling how E3 ligases are regulated is important because miscontrolled ubiquitylation can lead to disease. Cellular inhibitor of apoptosis (cIAP) proteins are E3 ligases that modulate diverse biological processes such as cell survival, proliferation, and migration. Here, we have solved the structure of the caspase recruitment domain (CARD) of cIAP1 and identified that it is required for cIAP1 autoregulation. We demonstrate that the CARD inhibits activation of cIAP1's E3 activity by preventing RING dimerization, E2 binding, and E2 activation. Moreover, we show that the CARD is required to suppress cell proliferation and migration. Further, CARD-mediated autoregulation is also necessary to maximally suppress caspase-8-dependent apoptosis and vascular tree degeneration in vivo. Taken together, our data reveal mechanisms by which the E3 ligase activity of cIAP1 is controlled, and how its deregulation impacts on cell proliferation, migration and cell survival.Molecular cell 06/2011; 42(5):569-83. · 14.61 Impact Factor
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ABSTRACT: NALP3 inflammasome, composed of the three proteins NALP3, ASC, and Caspase-1, is a macromolecular complex responsible for the innate immune response against infection with bacterial and viral pathogens. Formation of the inflammasome can lead to the activation of inflammatory caspases, such as Caspase-1, which then activate pro-inflammatory cytokines by proteolytic cleavage. The assembly of the NALP3 inflammasome depends on the protein-interacting domain known as the death domain superfamily. NALP3 inflammasome is assembled via a pyrin domain (PYD)/PYD interaction between ASC and NALP3 and a caspase recruitment domain/caspase recruitment domain interaction between ASC and Caspase-1. As a first step toward elucidating the molecular mechanisms of inflammatory caspase activation by formation of inflammasome, we report the crystal structure of the PYD from NALP3 at 1.7-Å resolution. Although NALP3 PYD has the canonical six-helical bundle structural fold similar to other PYDs, the high resolution structure reveals the possible biologically important homodimeric interface and the dynamic properties of the fold. Comparison with other PYD structures shows both similarities and differences that may be functionally relevant. Structural and sequence analyses further implicate conserved surface residues in NALP3 PYD for ASC interaction and inflammasome assembly. The most interesting aspect of the structure was the unexpected disulfide bond between Cys-8 and Cys-108, which might be important for regulation of the activity of NALP3 by redox potential.Journal of Biological Chemistry 08/2011; 286(45):39528-36. · 4.65 Impact Factor