The NLRP12 Pyrin Domain: Structure, Dynamics, and Functional Insights

Abstract

The initial line of defense against infection is sustained by the innate immune system. Together, membrane-bound TLR and cytosolic NLR receptors play key roles in the innate immune response by detecting bacterial and viral invaders as well as endogenous stress signals. NLRs are multi-domain proteins with varying N-terminal effector domains that are responsible for regulating downstream signaling events. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP12 (NLRP12 PYD) determined using NMR spectroscopy. NLRP12 is a non-inflammasome NLR that has been implicated in the regulation of TLR-dependent NF-κB activation. NLRP12 PYD adopts a typical 6-helical bundle death domain fold. By direct comparison with other PYD structures, we identified hydrophobic residues that are essential for the stable fold of the NLRP PYD family. In addition, we report the first in vitro confirmed non-homotypic PYD interaction between NLRP12 PYD and the pro-apoptotic protein FAF-1, which links the innate immune system to apoptotic signaling. Interestingly, all residues that participate in this protein:protein interaction are confined to the α2-α3 surface, a region of NLRP12 PYD that differs most between currently reported NLRP PYD structures. Finally, we experimentally highlight a significant role for tryptophan 45 in the interaction between NLRP12 PYD and the FAF-1 UBA domain.

Structure of the Absent in Melanoma 2 (AIM2) Pyrin Domain Provides Insights into the Mechanisms of AIM2 Autoinhibition and Inflammasome Assembly

“…To date, the structures of eight human PYDs and two mouse PYDs have been experimentally characterized, largely through nuclear magnetic resonance (NMR) spectroscopy. These include the PYDs from human ASC (Protein Data Bank codes 1UCP (25) and 2KN6 (26)), NLRP1 (Protein Data Bank code 1PN5) (27), NLRP3 (Protein Data Bank code 3QF2) (28), NLRP4 (Protein Data Bank code 4EWI) (29), NLRP7 (Protein Data Bank code 2KM6) (30), NLRP12 (Protein Data Bank code 2L6A) (31), and POP1/ASC2 (Protein Data Bank code 2HM2) (32) and mouse NLRP10 (Protein Data Bank code 2DO9) as well as the PYHIN family members myeloid cell nuclear differentiation antigen (Protein Data Bank code 2DBG; human) and p205b (Protein Data Bank code 2YU0; mouse). Interestingly, all known PYD structures have a uniquely short ␣3 helix compared with other DD superfamily members (33).…”

ASC Pyrin Domain Self-associates and Binds NLRP3 Protein Using Equivalent Binding Interfaces

“…However, some structural differences between the subfamilies are observed within this common fold, including different charge distributions throughout the protein surface, diverse locations of hydrophobic patches, and the different lengths of certain ␣-helices. These differences are thought to lead to homotypic binding specificity (5), albeit certain heterotypic interactions are also known with proposed inhibitory roles in apoptotic and inflammatory pathways (1,6).…”

“…The PYD domain is characterized at the structural level by a large loop connecting helices 2 and 3, with the latter being particularly short (6,17,(32)(33)(34)(35)(36)(37)(38). Despite the abundant structural information reported for PYD domains (the three-dimensional structures of 10 different PYD domains have been deposited in the PDB) (6,17,29,(32)(33)(34)(35)(36)(37)(38), our understanding on the ASC⅐NLRP3 PYD homotypic interaction is very limited.…”

“…Despite the abundant structural information reported for PYD domains (the three-dimensional structures of 10 different PYD domains have been deposited in the PDB) (6,17,29,(32)(33)(34)(35)(36)(37)(38), our understanding on the ASC⅐NLRP3 PYD homotypic interaction is very limited. Several models for this interaction have been proposed based on the NLRP3 crystallographic structure and mutational data (4,16), in addition to the recent semi-atomic structure characterization of ASC PYD mature fibril assemblies by cryo-EM and solid-state NMR (18,39).…”

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.…”