Three-dimensional Structure of the NLRP7 Pyrin Domain

ASC PYD

ASC PYD was cloned into pET28 (Novagen). The plasmids were transformed into Escherichia coli BL21 (DE3) cells. The cultures were grown to an A₆₀₀ of ∼0.8 at 37 °C, and expression was induced by the addition of 0.4 mm isopropyl β-d-thiogalactopyranoside. Proteins were expressed for 5 h at 25 °C. Cells were harvested by centrifugation, resuspended in 30 ml of buffer (50 mm Tris, pH 8.0, 100 mm NaCl), transferred to a 50-ml Falcon tube, and stored at −20 °C overnight. For purification, the cells were lysed by sonication (5 pulses of 70 W for 35 s) on ice. Immediately following sonication, 5 μl of DNase (1 unit/μl) was added to the lysed sample, and it was incubated for 20 min on ice. The cell debris was harvested by centrifugation (50,000 × g, 20 min, 4 °C). The ASC-containing pellet was resuspended in 25 ml of urea buffer (50 mm Tris, pH 8.0, 8 m urea) and incubated overnight at room temperature. Following centrifugation (50,000 × g, 20 min, 4 °C), the supernatant containing denatured ASC was loaded onto a nickel-nitrilotriacetic acid column. The column was then washed with 20 ml of wash buffer 1 (20 mm Tris, pH 8.0, 6 m urea, 5 mm imidazole) followed by 20 ml of wash buffer 2 (20 mm Tris, pH 8.0, 1 m NaCl, 20 mm imidazole). The protein was eluted three times with 1.2 ml of elution buffer (50 mm citric acid, pH 4.0, 1 m NaCl, 5 mm dithiothreitol). The purity of ASC was verified by SDS-PAGE, and its folded state was verified by NMR spectroscopy.

FAF-1 FID

The FAF-1 FID domain (residues 1–180) was cloned into RP1B (39). Freshly transformed E. coli BL21-Codon-Plus (DE3)-RIL (Stratagene) cells were grown at 37 °C to an A₆₀₀ of ∼0.6, and expression was induced with 1 mm isopropyl β-d-thiogalactopyranoside. The proteins were expressed for 18 h at 18 °C. The cultures were harvested by centrifugation, and the cell pellets were stored at −80 °C. The cell pellets were resuspended in lysis buffer (50 mm Tris-HCl, pH 8.0, 5 mm imidazole, 500 mm NaCl, 0.1% Triton X-100) supplemented with a Complete EDTA-free protease inhibitor tablet (Roche Applied Science). The cells were lysed by high pressure homogenization (Avestin C-3 Emulsiflex), and the cell debris was removed by centrifugation (50,000 × g, 40 min, 4 °C). The clarified lysate was loaded onto a His-Trap HP Column (GE Healthcare), and the protein was eluted with a 5–500 mm imidazole gradient. Fractions containing the FAF-1 FID domain were pooled, incubated with TEV protease to remove the N-terminal Thio₆His₆ tag, and dialyzed against fresh buffer (20 mm Tris-HCl, pH 7.5, 200 mm NaCl). The cleaved sample, containing the now untagged FAF-1 FID domain, was loaded onto a nickel-nitrilotriacetic acid column (Invitrogen), from which it eluted in the flow through. For the final purification step, the protein was loaded onto a Superdex 75 26/60 size exclusion column (GE Healthcare) equilibrated with 20 mm sodium phosphate, pH 6.0, 200 mm NaCl, 0.5 mm tris(2-carboxyethyl)phosphine. Fractions containing the pure FAF-1 FID domain, as confirmed by SDS-PAGE, were pooled and concentrated.

Protein Expression and Purification

Numerous NLRP7 PYD constructs with different C-terminal lengths were tested for overexpression. NLRP7_1–96 (NLRP7 PYD) showed the highest overexpression of soluble and folded protein and was subsequently used for all structural studies. NLRP7 PYD is a monomer in solution as verified by size exclusion chromatography, where a sharp peak containing NLRP7 PYD corresponding to a molecular mass of a ∼12 kDa was detected, as identified by SDS-PAGE analysis. The monomeric state of NLRP7 PYD was further confirmed by dynamic light scattering, which showed an R_h of 1.77 nm, an expected radius for a 12.7-kDa globular protein. The ASC PYD domain was expressed and refolded as described under “Experimental Procedures.” The FAF-1 FID domain (residues 1–180) was produced in a similar manner to the NLRP7 PYD. The FAF-1 FID domain is a monomer in solution as verified by size exclusion chromatography.

NLRP7 PYD Structure Determination and Analysis

The NLRP7 PYD three-dimensional structure adopts a typical death domain fold (Fig. 1, A and B), which is comprised of six α-helices, α1–α6, that fold into an anti-parallel α-helical bundle. The α-helical bundle tightly packed around a central hydrophobic core comprised of residues Leu⁶, Leu¹⁰, Leu¹³, Leu¹⁴, and Leu¹⁷ from α1; Leu²², Phe²⁵, and Leu²⁹ from α2; Leu⁵⁴ and Leu⁵⁸ from α4; Ile⁶⁷, Ile⁷⁴, and Leu⁷⁵ from α5; and Leu⁸³ and Ala⁸⁷ from α6 (Fig. 1C). In addition, α3 is stabilized by an additional hydrophobic cluster anchored by residues Leu²⁹, Trp³⁰, and Phe³² from α2; Leu³⁴ and Leu³⁸ from the α2-α3 loop (residues 30–35); and Thr⁴¹ and Trp⁴³ from α3, which position α3 with α2 (Fig. 1D). Short linker regions, including residues 18–20, 30–35, 48–50, 61–64, and 79–82 connect the six helices (Fig. 1B). All of the loops are well defined by dense NOE networks that connect the loops with the six α-helices (Fig. 1A).

NLRP7 PYD and the PYDs of NLRP1, ASC, and ASC2 Show Distinct Structural Features

The PYD domains of NLRP7, NLRP1 (24% sequence identity; Protein Data Bank accession code 1PN5) (34), ASC (27% sequence identity; Protein Data Bank accession code 1UCP) (31, 57), and ASC2 (25% sequence identity; Protein Data Bank accession code 2HM2) (32) only share modest primary sequence similarity (Fig. 2). ASC2 (also referred to as POP1 and ASCI) functions as a dominant negative inhibitor of ASC and other PYD-containing proteins involved in NF-κB and caspase-1 activation. The structure of mouse NLRP10 has also been determined (Protein Data Bank accession code 2DO9), but because the structure determination of the mNLRP10 PYD has not been published and no dynamics data have been reported for this domain, we refer to our comparison with the mNLRP10 PYD in supplemental Figs. S1–S3.

Despite the low overall sequence similarity, multiple hydrophobic residues that are essential for the formation of the PYD hydrophobic core are highly conserved, not only among these PYD domains but also across the entire NLRP1–14 family. These residues include Leu¹⁴, Leu¹⁷, Phe²⁵, and Leu⁵⁸. Although the hydrophobic core of PYDs is conserved, the architecture of the helices, especially helix α3 and the loop connecting helices α2 and α3, differs significantly (Fig. 3A). The α2-α3 loop and helix α3 are well ordered in NLRP7 PYD. The well ordered α2-α3 loop in NLRP7 is strongly anchored by six hydrophobic residues (Trp³⁰, Phe³², Leu³⁴, Leu³⁸, Thr⁴¹, and Trp⁴³; Fig. 3B, left panel). An α-helical turn can be identified for residues Val³⁷–Lys⁴⁰ in NLRP7 PYD. Similarly, the α2-α3 loops of ASC and ASC2 are also well ordered and adopt a similar structure, although helix α3 in ASC2 is shorter by one turn. Four or three hydrophobic residues anchor the α2-α3 loop in ASC and ASC2, respectively (Leu³⁸, Leu³², Tyr³⁶, and Ile³⁹ in ASC; Leu³², Phe³⁶, and Ile³⁹ in ASC2; Fig. 3B, middle panels). In addition, hydrophobic and polar interactions between helix α3 and α4 (Fig. 3, C and D) further stabilize helix α3 in the NLRP7, ASC, and ASC2 PYDs.

In contrast, no α2-α3 loop hydrophobic residues or hydrophobic contacts between helices α2, α3, and α4 can be identified in the NLRP1 PYD that could contribute toward any conformational stability of this loop (Fig. 3, B and D, right panels). As a result, helix α3 is missing in the NLRP1 PYD structure, and the α2-α3 loop is highly dynamic, as shown by dynamics NMR measurements (34), and extended toward helix α4.

To further quantify these differences in hydrophobic interactions, the hydrophobic surface buried by helices α2 and α3 and the α2-α3 loop was calculated using GetArea1.1 (58): NLRP1 PYD, 1763 Ų; ASC2 PYD, 2026 Ų; ASC PYD, 2199 Ų; and NLRP7 PYD, 2266 Ų. These calculations show a large 25% increase in hydrophobic surface burial between the NLRP1 and NLRP7 PYDs (this increase in hydrophobicity is highlighted by a black arrow in Fig. 3B).

The significant increase of the number of hydrophobic residues in the α2-α3 loop and helix α3 from NLRP1 to NLRP7 allows for the formation of an exceedingly stable second hydrophobic cluster in the NLRP7 PYD (Fig. 1D). These hydrophobic residues are also responsible for the differential flexibility of the α2-α3 loop (described in the next section), as well as the formation of helix α3. We used the program AGADIR (59) to predict the helical behavior of the residues between the C termini of helices α2 and α3. The calculations show very low propensities for helix formation (<1%), confirming that the presence of helix α3 in the structures of NLRP7, ASC, and ASC2 PYDs is due primarily to tertiary contacts within the proteins.

Thus, although the conserved central hydrophobic cores of PYDs are expected to lead to a similar fold for helices α1, α2, and α4–α6 (Figs. 1C and 2) for all PYDs, further differences in the conformations of the α2-α3 loop and helix α3 are expected in other PYDs whose structures have not yet been determined. Therefore, the amino acid composition and, as a consequence, the structural and dynamics behavior of the α2-α3 loop and helix α3 likely function as key regulators of the differential signaling by the NLRP receptor family.

NLRP7 PYD Auto- and Cross-correlated Motion Analysis

To further understand the differences between the PYDs, we measured auto-correlated ¹⁵N-relaxation data: ¹⁵N{¹H}-NOE, ¹⁵N longitudinal (R₁), and transverse (R₂) relaxation rates. Similar measurements were reported for the ASC2 and NLRP1 PYD domains, as well as for full-length ASC (which includes both the N-terminal PYD and the C-terminal CARD domain). Comparison of the ¹⁵N{¹H}-NOE data, which reports on fast time scale (ps to ns) backbone motions, is shown in Fig. 4A. Although the α2-α3 loop in the PYDs of NLRP7, ASC, and ASC2 exhibit rigidities nearly identical to their neighboring α-helices, the α2-α3 loop in the NLRP1 PYD shows substantially increased dynamics. The observed increase in flexibility is most likely a result of the absent second hydrophobic cluster in the NLRP1 PYD.

A R₂/R₁ ratio plot identified that NLRP7 PYD residues Trp³⁰, Leu³⁴, Val³⁷, Glu³⁹, Thr⁴¹, Trp⁴³, and Ser⁴⁴, which are located in the α2-α3 loop, have increased R₂/R₁ ratios (supplemental Fig. S4). This shows that these residues undergo motions on a slower time scale than those of the rest of the protein. Indeed, in a model-free relaxation analysis (τ_c = 6.4 ns), these residues required the inclusion of a R_ex term to accurately model the data (supplemental Fig. S4). To further confirm the dynamics of this loop, we recorded R₂ rates at two static fields (11.7 and 18.8 T), as well as cross-correlation rates between the ¹⁵N chemical shift anisotropy and ¹⁵N-¹H dipole-dipole interactions (η_xy) (supplemental Fig. S4). The cross-correlation rates were used to calculate the exchange contribution using R_ex = R₂ − κη_xy (Fig. 4B), which confirmed the R_ex contribution for the aforementioned residues. Using on-resonance R_1ρ measurements (radio-frequency spin lock = 2 kHz), we determined that the chemical exchange is in the microsecond time regime (supplemental Fig. S4).

Interestingly, two proline residues, Pro³³ and Pro⁴², flank the α2-α3 loop of the NLRP7 PYD. Pro⁴² is conserved in the PYDs of NLRP7, ASC, ASC2, and NLRP1. However, Pro³³ is only conserved in NLRP7, ASC, and ASC2 PYDs; it is not present in NLRP1 PYD. A number of cross-peaks from amino acids N- and C-terminal to these prolines show small satellite peaks in the two-dimensional ¹H,¹⁵N HSQC, indicating a minor conformation, most likely caused by a cis/trans-isomerization of Pro³³ and Pro⁴² (supplemental Fig. S5). By comparison of the peak intensities, the minor conformation is estimated to be present at a level of ∼10%. A similar 10% cis/trans-proline isomerization was identified for Pro⁴² in NLRP1 PYD.

The NLRP7, NLRP1, ASC, and ASC2 PYDs Have Distinct Electrostatic Surfaces

Electrostatic surface charge plays a major role in the formation and regulation of death domain protein-protein interactions (60). Initial work on the RAIDD CARD, as well as caspase-9 and Apaf-1 CARD domains, showed the importance of highly charged surface patches for the specific interaction of CARD domains (35, 61). A similar mode of electrostatic interaction was also identified for the PYD-PYD interaction of ASC PYD and ASC2 PYD (37). Critically, the PYD-PYD interaction between NLRP1 PYD and ASC PYD and, in turn, the CARD-CARD domain interaction between the ASC CARD and caspase-1 CARD domains are essential for the formation of the inflammasome. Only NLRP1, NLRP3, and NLRC4 have been identified so far to form inflammasomes with clear physiological roles (14).

The electrostatic surface of ASC PYD is distinctively two-faced. A highly positively charged surface is formed near helices α2 and α3, and a negatively charged surface is observed near helices α1 and α4 (Fig. 5, A and D, respectively). ASC2 PYD binds via its positively charged helices α2 and α3 surface to the negative charged surface formed by residues from helices α1 and α4 of ASC PYD (comprised of residues Lys²¹, Lys²², Arg³⁸, and Arg⁴¹ and residues Asp⁶, Asp¹⁰, Glu¹³, Asp⁴⁸, Asp⁵¹, and Asp⁵⁴ from ASC2 and ASC, respectively). Thus oppositely charged electrostatic surfaces are important for mediating these PYD-PYD interactions (37).

Fig. 5 compares the electrostatic surface of NLRP7 PYD with those of ASC, ASC2, and NLRP1. We used the Protein Interaction Property Similarity Analysis server (62) to quantify the similarities between the electrostatic surfaces of the PYDs from NLRP1, NLRP7, ASC, and ASC2. The Protein Interaction Property Similarity Analysis server uses the University of Houston Brownian Dynamics program to calculate the electrostatic potential of a protein. Pairwise calculations comparing the NLRP7 PYD to the PYDs of ASC, ASC2, and NLRP1 were used to assess overall electrostatic similarity, where 0 indicates identical electrostatic surfaces, and 2 indicates completely different electrostatic surfaces. Based on an overall electrostatic distance, NLRP7 PYD is most similar to ASC PYD (electrostatic distance, 1.115) followed by NLRP1 PYD (electrostatic distance, 1.145) and ASC2 PYD (electrostatic distance, 1.293), although none of the other PYDs are identical/highly similar to the NLRP7 PYD (i.e. an electrostatic distance between 0 and 0.75).

In fact, a closer look at specific surfaces, such as the α2 and α3 and α1 and α4 interaction surfaces (hereafter referred to as interaction patches) illustrates distinct differences in the electrostatic surfaces of these PYDs. ASC2 PYD has the most similar interaction patch electrostatic surface to ASC PYD. Conversely, the interaction patch electrostatic surfaces of NLRP1 PYD and NLRP7 PYD are highly dissimilar. NLRP1 PYD has only a small positively charged interaction patch, similar to ASC PYD and ASC2 PYD, formed mainly by Lys²² on helix α2. This positively charged patch is completely absent on NLRP7 PYD. Indeed, this surface of the NLRP7 PYD is nearly completely hydrophobic with small negatively charged patches. Conversely, the negative charges on opposing surfaces, especially Glu¹⁵ and Glu⁵⁶ from helices α1 and α4, respectively, are much more similar between the PYDs of NLRP7, ASC, and ASC2. Indeed, the NLRP1 PYD has the least negatively charged electrostatic surface at this interaction patch.

The PYDs of ASC and ASC2 have structurally opposed positively and negatively charged electrostatic interaction patches. Critically, these oppositely charged patches have also been shown to be essential for mediating homotypic protein-protein interactions between these PYDs. These electrostatic surfaces of these interaction patches are significantly different for the PYDs of NLRP1 and NLRP7, with the positively charged surface patch necessary for a potential ASC PYD interaction completely missing in NLRP7 PYD. This observation predicts that a protein-protein interaction between the PYDs of ASC and NLRP7 will not occur. To confirm this hypothesis, we experimentally tested the interaction of the NLRP7 PYD with ASC PYD using NMR spectroscopy. Unlabeled ASC PYD was titrated into ¹⁵N-labeled NLRP7 PYD. No chemical shift changes in the NLRP7 PYD two-dimensional ¹H,¹⁵N HSQC spectrum were identified upon titration with ASC PYD (1:1 molar ratio), confirming our hypothesis that the NLRP7 PYD does not bind directly to the ASC PYD (supplemental Fig. S6).

NLRP7 PYD Does Not Interact Directly with FAF-1

FAF-1 is a multidomain protein and a conserved pro-apoptotic factor that also has a central role in the regulation of NF-κB. Recently, it was reported that NLRP3, NLRP7, and NLRP12 interact with FAF-1 via its Fas-interacting domain (FID) (19). The reported interaction studies showed that the interaction must be mediated by residues 1–180 of the FAF-1 FID domain. The FAF-1 FID domain consists of an N-terminal UBA domain, known to interact with polyubiquitinated proteins, as well as a C-terminal adjacent UB1, which has a typical ubiquitin fold and is implicated in the inhibition of NF-κB signaling.

We used NMR spectroscopy to experimentally test the potential direct interaction of NLRP7 PYD with the FAF-1 FID domain. This is of interest because it would be the first nonhomotypic PYD interaction ever confirmed in vitro. The unlabeled FAF-1 FID domain was titrated into ¹⁵N-labeled NLRP7 PYD. No chemical shift changes in the NLRP7 PYD two-dimensional ¹H,¹⁵N HSQC spectrum were identified upon titration with FAF-1 FID (1:1 and 1:10 molar ratio; supplemental Fig. S7), demonstrating that the NLRP7 PYD does not interact directly with the FAF-1 FID domain.