Molecular Basis for Specific Recognition of Bacterial Ligands by NAIP/NLRC4 Inflammasomes

Abstract

SUMMARY NLR (nucleotide-binding domain [NBD]- and leucine-rich repeat [LRR]-containing) proteins mediate innate immune sensing of pathogens in mammals and plants. How NLRs detect their cognate stimuli remains poorly understood. Here, we analyzed ligand recognition by NAIP (NLR Apoptosis Inhibitory Protein) inflammasomes. Mice express multiple highly related NAIP paralogs that recognize distinct bacterial proteins. We analyzed a panel of 43 chimeric NAIPs, allowing us to map the NAIP domain responsible for specific ligand detection. Surprisingly, ligand specificity was mediated not by the LRR domain, but by an internal region encompassing several NBD-associated α-helical domains. Interestingly, we find that the ligand specificity domain has evolved under positive selection in both rodents and primates. We further show that ligand binding is required for the subsequent co-oligomerization of NAIPs with the downstream signaling adaptor NLRC4 (NLR family, CARD-containing 4). These data provide a molecular basis for how NLRs detect ligands and assemble into inflammasomes.

The NLRC4 inflammasome: The pieces of the puzzle are falling into place

“…Yet, how NAIPs recognized their ligands remained unknown. As mentioned earlier, mice have several NAIPs with high diversity among different inbred strains [25]. Exploiting this high variability, an extensive mutagenesis study of more than 40 chimeric proteins enabled the mapping of the parts of the NAIP molecules responsible for the recognition of and response to bacterial components (Fig.…”

“…Exploiting this high variability, an extensive mutagenesis study of more than 40 chimeric proteins enabled the mapping of the parts of the NAIP molecules responsible for the recognition of and response to bacterial components (Fig. 1D) [25]. Switching segments between points, mostly located within regions of high sequence identity among NAIPs, enabled the mapping of the PAMP specificity domain to regions comprising of HD1, WHD, and HD2 and the unannotated segment between HD2 and LRR of NAIP5 for flagellin, and HD1, WHD, and HD2 of NAIP2 for the rod protein of T3SS (Fig.…”

“…1D). Tenthorey et al [25] also showed that the LRR domain is not the domain that determines the specificity of NAIPs and is probably not necessary for PAMP recognition at all, unless it binds a motif common to both flagellin and T3SS proteins. This is in contrast to a well-established LRR domain function in Toll-like receptors and also in plant NLR proteins [26,27], but does not contradict the repressive role of the LRR domain [13], Approximate locations of insets are depicted on the main structure.…”

“…activation ( Fig. 3B) proposes that NLRC4 activation is initiated by respective NAIPs recognizing bacterial ligands through the HD1-WHD-HD2 region [25]. The ligand induces conformational change, exposing NAIP's nucleating surface, which binds to NLRC4 via its receptor surface.…”

Inflammasome assembly: The wheels are turning

“…Previous work showed that the NOD domain of NAIPs confers ligand binding and specificity [10], yet according to the new studies the same domain also forms the nucleating surface that initiates complex assembly. Since thus far the structure of inactive or activated NAIPs has not been reported, the structural basis underlying ligand-NOD domain interaction is unknown and the resulting conformational changes remain to determined.…”