Crystal structure of the Nod1 caspase activation and recruitment domain

Abstract

Nod-like receptors (NLRs), Nod1 and Nod2 are cytosolic detectors of pathogen associated molecular patterns (PAMPs). Nod1 is a three-domain protein, consisting of a caspase activation and recruitment domain (CARD), a nucleotide-binding oligomerization domain (NOD), and a leucine-rich repeat domain (LRR). The binding of PAMPs to the LRR results in the activation of signaling through homophilic CARD-CARD interactions. Several CARD structures have been determined, including a recent NMR structure of Nod1 CARD. In contrast to the reported NMR structure, the crystal structure reported here is a dimer, where the sixth helix is swapped between two monomers. While the overall structure is very similar to the known CARD structures, this is the first report of a homodimeric CARD structure. The ability of the CARD to exist in monomeric and dimeric forms suggests another level of regulation in the activation of NLR proteins.

Ubiquitin Regulates Caspase Recruitment Domain-mediated Signaling by Nucleotide-binding Oligomerization Domain-containing Proteins NOD1 and NOD2

“…His 6 -NOD1 CARD was affinity-purified over Talon Co 2ϩ -agarose as described previously (32). Imidazole-eluted protein was then purified by gel filtration over Superdex 75 equilibrated with GF buffer (25 mM sodium phosphate buffer, pH 7.0, with 25 mM NaCl and, depending on the experiment, 5 mM DTT).…”

“…Our previously determined crystal structure of NOD1 CARD showed that it formed helix-swapped homodimers, juxtaposing two cysteines (Cys-39) near each other (supplemental Fig. S1, A andD) (32). Indeed, NOD1 CARD in solution lacking added reducing agents readily forms dimers linked by a disulfide bond, and this dimeric form has been observed in a different crystal structure (48).…”

Polypeptide Modulators of Caspase Recruitment Domain (CARD)-CARD-mediated Protein-Protein Interactions

“…The NLR proteins utilize the CARD for binding to downstream signaling molecules through CARD-CARD interactions in order to ultimately initiate the innate immune and inflammatory responses (9,10). As a general organization, the CARD module is characterized by a Greek key of six antiparallel amphipathic closely packed ␣-helices (11)(12)(13). However, primary sequence alignments reveal a very low sequence identity (Ͻ20%), emphasizing that some critical conserved residues could mediate initial CARD-CARD electrostatic interactions and that the surrounding residues in this region would define the specificity of CARD-CARD interactions (8,12).…”

International Union of Basic and Clinical Pharmacology. XCVI. Pattern Recognition Receptors in Health and Disease

“…At the atomic level, structural data exist for the nucleotide-binding domain (NBD)-LRR of NLRC4 (Hu et al, 2013), the LRRs of NLRX1 and NLRP1, and the effector domains of NOD1 (CARD), NLRC5 (atypical CARD), NLRP1 (CARD and pyrin domain [PYD]) (Hiller et al, 2003), and NLRP3, 4, 7, 10, 12, and 14 (all PYD) (Coussens et al, 2007;Manon et al, 2007;Srimathi et al, 2008;Pinheiro et al, 2010Pinheiro et al, , 2011Bae and Park, 2011;Eibl et al, 2012Eibl et al, , 2014Su et al, 2013;Jin et al, 2013;Gutte et al, 2014). Although we still await the structure of an NLR in complex with either its ligand or a downstream signaling adaptor, these structures have provided important insight into the molecular functionality of NLR signaling regulation and transduction.…”

“…The solution structure of the NOD1 CARD is a monomer (Manon et al, 2007), but the crystal structure is a dimer, stabilized by an interprotomer disulfide between Cys39 residues, in which helix 6 of one protomer swaps with helix 1 of the other (Coussens et al, 2007;Srimathi et al, 2008). Interestingly the crystal structure of the NLRP14 PYD also shows a similar process of helix swapping (Eibl et al, 2014) (Fig.…”