Structure and Dynamics of ASC2, a Pyrin Domain-only Protein That Regulates Inflammatory Signaling

Abstract

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-B and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15 N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding ␣2-␣3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the ␣2-␣3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the ␣2-␣3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.Inflammatory caspases such as caspase-1 play an essential role in innate immune responses to infection by regulating the processing of pro-inflammatory cytokines interleukin-1␤ and interleukin-18 into their mature, secreted forms (1, 2). Tight regulation of the production of these cytokines is required to maintain the homeostasis of host tissues. Excessive interleukin-1␤ production and chronic inflammation are hallmarks of many autoimmune diseases that present both systemically and within the central nervous system, including rheumatoid arthritis and multiple sclerosis (3, 4).Similar to initiator caspases involved in apoptosis, the activation of inflammatory caspases requires their recruitment into a multiprotein signaling complex, which promotes dimerization and cleavage to produce the active enzyme via an induced proximity mechanism (2, 5). Recently, models for caspase-1 activation have been proposed whereby inflammatory stimuli promote the formation of molecular platforms referred to as inflammasomes (6). The NALP1 inflammasome induces the activation of both caspase-1 and caspase-5 through the formation of a complex that also contains the proteins NALP1 and ASC (7). Similarly, the NALP2/NALP3 inflammasome is involved in the activation of caspase-1 through the r...

Multiple Binding Sites on the Pyrin Domain of ASC Protein Allow Self-association and Interaction with NLRP3 Protein

“…5B). These data are consistent with a conserved binding mode of the ASC PYD with these (42), and NLRP3 (Protein Data Bank ID code 3QF2) (39) were aligned using PyMOL, and the residues important for interaction of the ASC PYD are indicated with acidic residues in red and basic residues in blue. C, surface electrostatic potential of the PYDs of ASC, POP1, and NLRP3 shown in the same orientation as B.…”

“…Because NLRP3 assembles into an oligomer (6), its interaction with ASC PYD is likely to be promoted by self-association because of increased contacts between self-associated ASC and the NLRP3 oligomer as in our model. However, because POP1 is a monomer (42), ASC selfassociation does not promote its interaction. The fact that overlapping sites on ASC PYD interact with POP1 and NLRP3 suggested that POP1 may inhibit interaction of ASC with NLRP3.…”

“…Specific point mutations were introduced into ASC PYD using the QuikChange site-directed mutagenesis approach (Stratagene). Full-length POP1 (residues 1-89) was subcloned into pET-21a (Novagen) for in vitro translation, and His 6 -tagged POP1 was expressed in E. coli as previously described (42). The Pyrin PYD (residues 1-92) was subcloned into pET-28b (Novagen) for expression with a C-terminal His 6 tag.…”

“…The structures of various PYDs in isolation have been determined, including ASC (37), NLRP1 (38), NLRP3 (39), NLRP7 (40), NLRP12 (41), POP1 (42), and murine NLRP10 (Protein Data Bank ID code 2DO9). Several PYDs including ASC, POP1, and NLRP3 have distinct positively and negatively charged surface patches, and this has led to the proposal that electrostatic interactions will be critical for PYD interactions (37,39,42). However, the interaction sites have not been systematically mapped for any PYDs.…”

Three-dimensional Structure of the NLRP7 Pyrin Domain

“…Understanding how NLRP7 mediates signal transduction at a molecular level may provide new therapeutic approaches to these diseases. Despite the abundance of PYDs in several protein families, structural information about PYDs is very limited (31)(32)(33)(34). Currently, only a few structures of effector domains involved in NLR signaling are known.…”