Mapping of POP1-binding Site on Pyrin Domain of ASC

Abstract

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an essential adaptor protein in the formation of a multiprotein complex that activates procaspase-1. ASC is also known as a modulator of NF-B activation pathways. ASC has a bipartite domain structure, consisting of an N-terminal pyrin domain (PYD) and a C-terminal caspaserecruitment domain. The PYD of ASC (ASC_PYD) is known to interact with various PYD-containing intracellular danger signal sensors and PYD-only proteins. Using purified proteins, we characterized the in vitro interaction of ASC_PYD with PYDonly protein 1 (POP1). POP1 specifically interacts with ASC_PYD with a dissociation constant of 4.08 ؎ 0.52 M but does not interact with Cryopyrin. NMR and mutagenesis experiments show that a negative electrostatic potential surface patch (EPSP) on ASC_PYD, consisting of the first (H1) and fourth (H4) helices, is essential in the interaction with POP1. A positive EPSP on POP1, consisting of the second (H2) and third (H3) helices, is a counterpart of this interaction. The interaction between ASC_PYD and POP1 is similar to the interaction between caspase recruitment domains of Apaf-1 and procaspase-9. In addition, we present evidence that conformational changes at the long loop of ASC_PYD between the H2 and H3 helices can affect its interaction with POP1. Based on our observations, we propose that the positive EPSP of ASC_PYD, including the H2 and H3 helices, may be the binding site for Cryopyrin, and the interaction with Cryopyrin may induce the dissociation of POP1 from ASC_PYD.Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC; also known as Pycard and TMS1) is a bipartite adaptor protein that plays a role in proinflammatory cytokine production as well as NF-B activation (1, 2). ASC consists of an N-terminal pyrin domain (ASC_PYD) 3 and a C-terminal caspase-recruitment domain (ASC_CARD).Accumulating evidence indicates that ASC_CARD is involved in the recruitment and activation of procaspase-1 (3, 4). ASC_PYD interacts with PYD-containing NALP proteins to initiate a multiprotein platform known as an inflammasome, where procaspase-1 is activated by the induced proximity mechanism (3, 4). The NALP proteins are characterized by a common structure, consisting of N-terminal PYD, an intermediate NATCH domain, and C-terminal leucine-rich repeats. The NALP proteins are expected to function as intracellular danger signal sensors (5, 6). Cryopyrin (also known as NALP3, PYPAF1, and CIAS1) is a relatively well studied member of the NALP proteins. Recent studies using Cryopyrin Ϫ/Ϫ mice indicate that this protein is pivotal to the interleukin-1␤ production by macrophages in response to bacterial RNA (7), certain Gram-positive bacteria (8), uric acid, calcium pyrophosphate crystals (9), cellular toxins (10), and some Toll-like receptor agonists (8). In addition, Cryopyrin and other NALP proteins, in particular NALP6 (also known as PYPAF5) and NALP12 (also known as Monarch and PYPAF7), induce NF-B activat...

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

“…However, a previous study using NMR spectroscopy to map the surface of ASC PYD that interacts with POP1 proposed a role for residues Asp-6, Glu-13, Asp-48, and Asp-D54 on the negative surface but did not find any evidence of a role for residues in the positively charged surface (49). Notably, an L25A mutant was used in this study to improve the solubility of ASC PYD (49). However, our in vitro binding data show that the L25A mutation diminishes interaction of ASC PYD with POP1 (supplemental Fig.…”

“…S5C); thus it is not surprising that only very small chemical shift changes (⌬␦ Ͻ 0.07 ppm) were detected upon titration of 15 N-labeled ASC PYD (L25A) with POP1. Interestingly, the same study (49) showed that mutagenesis of Lys-21 (K21A) and Arg-41 (R41W) in the positive surface also diminished interaction of ASC PYD with POP1. This observation was proposed to be via a transduced effect, whereby conformational changes at one surface are communicated to the other surface through the hydrophobic core.…”

“…Our data based on in vitro GST pull-down assays as well as co-immunoprecipitation in HEK 293T cells clearly demonstrate that mutation of residues on both the positive and negative surfaces of ASC PYD also disrupt interaction with POP1. However, a previous study using NMR spectroscopy to map the surface of ASC PYD that interacts with POP1 proposed a role for residues Asp-6, Glu-13, Asp-48, and Asp-D54 on the negative surface but did not find any evidence of a role for residues in the positively charged surface (49). Notably, an L25A mutant was used in this study to improve the solubility of ASC PYD (49).…”

Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

“…The integration of data from two structural methods, cryo-electron microscopy and solid-state NMR spectroscopy, opens perspectives for structural studies of inflammasomes and related molecular assemblies. difficult to crystallize (11)(12)(13). In particular, the solution structures of isolated human ASC pyrin domain (ASC-PYD) and ASC fulllength protein (ASC-FL) have been determined, showing that the PYD and the CARD, connected by a flexible linker, tumble independently in solution (14,15).…”

Three-dimensional Structure of the NLRP7 Pyrin Domain

“…This has subsequently inspired speculation about the possibility that large conformational changes might occur upon PYD-PYD binding. Canonical PYD-PYD and CARD-CARD (35)(36)(37) interactions are driven primarily by strong electrostatic interactions and likely stabilized by the subsequent formation of hydrophobic interactions. For example, based on the total buried surface area in the APAF-1 CARD-caspase-9 CARD complex (35), it was estimated that 70% of the interaction was due to electrostatic interactions, and 30% was due to hydrophobic interactions.…”