Structural and Functional Analysis of the NLRP4 Pyrin Domain

Abstract

NLRP4 is a member of the nucleotide-binding and leucine-rich repeat receptor (NLR) family of cytosolic receptors and a member of an inflammation signaling cascade. Here, we present the crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution. The NLRP4 PYD is a member of the death domain (DD) superfamily and adopts a DD fold consisting of six α-helices tightly packed around a hydrophobic core, with a highly charged surface that is typical of PYDs. Importantly, however, we identified several differences between the NLRP4 PYD crystal structure and other PYD structures that are significant enough to affect NLRP4 function and its interactions with binding partners. Notably, the length of helix α3 and the α2−α3 connecting loop in the NLRP4 PYD are unique among PYDs. The apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein whose interactions with a number of distinct PYDs are believed to be critical for activation of the inflammatory response. Here, we use co-immunoprecipitation, yeast two-hybrid, and nuclear magnetic resonance chemical shift perturbation analysis to demonstrate that, despite being important for activation of the inflammatory response and sharing several similarities with other known ASC-interacting PYDs (i.e., ASC2), NLRP4 does not interact with the adaptor protein ASC. Thus, we propose that the factors governing homotypic PYD interactions are more complex than the currently accepted model, which states that complementary charged surfaces are the main determinants of PYD–PYD interaction specificity.

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

“…It is well known that ASC PYD solubility is too low at neutral pH for conducting NMR experiments (29,32). Therefore, previous NMR attempts to characterize ASC PYD interactions used an L25A mutant that is soluble at the typical NMR concentrations (27,37). Unfortunately, this mutation has been shown to diminish the binding capabilities of the protein (see below).…”

“…Electrostatic interactions have been traditionally considered as carrying most of the weight with PYD⅐PYD complex formation (16,18,37,41). As a result, information on the importance of apolar or hydrophobic interactions is scarce.…”

“…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.…”