Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein

VSports - Protein Production, Crystallization, and Structure Determination.

Initially, GSDMB_C (Glu244–Ser411) was produced as a C-terminal His-tagged protein. The circular dichroism (CD) spectrum of this protein showed a molecule containing predominantly α-helices, consistent with the secondary structure prediction. The ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) NMR spectrum of GSDMB_C revealed well-dispersed peaks accounting for most of the amino acids. Nevertheless, this GSDMB_C failed to crystallize. Therefore, we produced the GSDMB_C linked to maltose-binding protein (MBP) using the pMALX vectors (34). These recombinant proteins contain the interdomain linker region (Met220–Lys240) that was systematically truncated by 4- to 5-aa segments. Several recombinant proteins were purified to homogeneity, but only the longest variant (MBP followed by GSDMB Met220–Ser411, MBP-GSDMB_C) yielded crystals. To test the structural impact of disease-associated polymorphisms, four possible variants were crystallized: the two variants found in the population (Gly299:P306 and Arg299:S306) and two alternative combinations that are not observed in the population (Arg299:P306 and Gly299:Ser306). However, the crystals of the MBP-GSDMB_C Arg299:Pro306 variant diffracted X-rays poorly (∼7 Å) for analysis. Intriguingly, each variant crystallized in a different space group, with a different number of molecules in the asymmetric unit (Table 1). Together, there are 16 copies of GSDMB_C molecules, each exposed to a different crystal environment. The availability of multiple GSDMB_C molecules increases confidence in the structural analyses.

Overall Structure of the MBP-GSDMB Fusion Protein.

In each crystal structure, the MBP-GSDMB_C molecules pack in pairs using both crystallographic and noncrystallographic twofold symmetry axes. Fig. 2A presents such a pair in the asymmetric unit of the MBP-GSDMB_C Arg299:Ser306 structure (PDB ID code 5TIB), which form an interface area of 1,250 Ų, calculated using PISA (35). The corresponding five pairs of the Gly299:Pro306 variant (PDB ID code 5TJ2) and two pairs of the Gly299:Ser306 variant (PDB ID code 5TJ4) molecules form averaged interface areas of 1,588 Ų and 1,164 Ų, respectively. There is no interaction between the two GSDMB_C domains, consistent with the monomeric GSDMB_C eluted from the Superdex 200 size exclusion column. Likewise, the full-length His₆-GSDMB (Gly299:Pro306 variant) also eluted predominantly as monomers. All 16 GSDMB_C domains in the three crystal structures exhibited the same overall fold. The root-mean-square deviations (rmsds) of C^α positions of the 10, 4, and 2 molecules in the asymmetric units of the MBP-GSDMB_C Gly299:Pro306, Gly299:Ser306 and Arg299:Ser306 variants were 0.4 to 0.6 Å.

Ensuing the MBP’s C-terminal α-helix, a three-alanine linker engineered in the pMALX vector elongates this α-helix (34). The following five GSDMB_C linker residues (Met220–Asp224) further extend the MBP α-helix (Fig. 2), apparently influenced by the presence of MBP. Thus, these five residues may not necessarily form an α-helix in the context of full-length GSDMB. The remaining GSDMB interdomain linker (Ile225–Arg247) adopts a flexible conformation with segments that lack associated electron density, as depicted by a blue dotted line in Fig. 2B. Inspection of all 16 molecules in the three crystal structures shows that the extent of linker conformational order/disorder correlates with different intermolecular contacts. Only three molecules in the MBP-GSDMB_C Gly299:Pro306 structure have well-resolved electron density over the entire linker region (molecules B, E and J; PDB ID code 5TJ2). The markedly different conformations of these resolved linkers underscore the apparent flexibility of this region, which would be a useful property for regulating the activity of GSDMB by possibly altering the relative orientation of the two domains.

The flexible linker is followed by the GSDMB_C core structure (Asn248–Pro406), exhibiting a bundle of 7 α-helices (α5–α11) (Figs. 1 and 2). The helical core contains another disordered loop connecting the last two helices, α10 and α11 (Glu367–Tyr382), as depicted by a red dotted line in Fig. 2B. Again, although two versions of this loop could be fully traced in the electron density of the MBP-GSDMB_C Gly299:Pro306 structure (molecules A and C; PDB ID code 5TJ2), these defined loops contain no secondary structure elements and adopt different conformations. Another loop within the core GSDMB_C connects the α7 and α8 helices (Arg299–Val322) shown at the bottom left in Fig. 2. This loop contains the two polymorphism amino acids at positions 299 and 306. All 16 versions of this loop contain a five-residue α-helix (labeled α′), spanning residues Pro309 to Ser313. However, molecules that contain a Ser at position 306 (MBP-GSDMB_C Arg299:Ser306 and Gly299:Ser306) adopt an additional four-residue helical turn at residues Met303–Ser306 between the α7 and α′ helices. Loops that contain a Pro at position 306 do not form this helical turn.

The seven-helix bundle topology of GSDMB_C and the related Gsdma3 topology (PDB ID code 5B5R) are unusual. A DALI search (36) revealed a single structure exhibiting the same topology: that of the cytoplasmic domain of a putative membrane protein from Clostridium difficile 630 (PDB ID code 3KMI, determined by the Midwest Structural Genomic Consortium). The DALI program calculates a Z-score of 6.5 and a C^α position rmsd value of 4.5 Å for 112 of the 167 aligned residues, with 13% amino acid sequence identity (Fig. 3). Despite the common topologies, the superposed structures show substantial differences (Fig. 3). The helices of the GSDMB_C core are much shorter, two loops are significantly longer, and GSDMB_C has an additional N-terminal α-helix (α5, colored gray in Fig. 3). The Pfam database annotates this structure (PDB ID code 3KMI) as belonging to the C-terminal domain of the ArAE_1_C aromatic acid exporter family (Transporter Classification Database; ref. 37). Escherichia coli YhcP, a member of the ArAE_1_C family, has been characterized as an efflux pump that alleviates toxic effects of p-hydroxybenzoic acid on the metabolic balance of E. coli (38). Taken together, the structural relationship seems too remote to derive conclusions about the function of GSDMB.

Impact of Amino Acid Residues 299 and 306 on the Structure.

Of the human genomes sequenced by the 1000 Genomes Project (33), the GSDMB polymorphisms encoding the alleles containing either Gly299:Pro306 or Arg299:Ser306 are present at ∼50% frequency in the general population (22). The GSDMB SNPs associated with inflammatory disease risks encode the Arg299:Ser306 variant. Fig. 4 compares the conformations of the loop region that carries the pairs of polymorphism residues. Loops containing Pro306 exhibit a wide range of backbone conformations (Fig. 4A). In contrast, loops containing Ser306 (MBP-GSDMB_C Arg299:Ser306 and Gly299:Ser306) exhibit a helical turn preceding Ser306 and a tight range of loop conformation (Fig. 4B). The backbone conformation close to residue 299 is similar, regardless of the presence of a glycine or an arginine in all 16 GSDMB_C molecules (Fig. 4B). However, the presence or absence of a positive charge, when position 299 is occupied by an Arg or a Gly, would impact the surface electrostatics in the vicinity. Modeling a Pro in the place of Ser at position 306 illustrates why a proline side chain cannot be accommodated in the context of the preceding helical turn (Fig. 4C). The model shows short contacts (<1.5 Å) between both the C^γ and C^δ atoms of the proline to the backbone oxygen atom of Met303. Apparently, the accommodation of the proline side chain at position 306 is accompanied by the loop adopting extensive conformational flexibility (Fig. 4A).

Unlike monogenic diseases, caused by high-penetrance SNPs in single genes, complex-trait diseases are associated with multiple low-penetrance SNPs in multiple genes. Because of linkage disequilibrium, most of the SNPs present in a genome are actually not disease-causative. The GWAS results highlighted the complexity of SNP patterns emerging from the genomic sequences of large populations and underscored the challenges of identifying truly disease-causative SNP candidates. The current study provides structural perspective for possible disease risk mechanisms attributed to candidate SNPs. In addition to the strikingly different conformations of the GSDMB Arg299:Ser306 and Gly299:Pro306 containing loops, a change from a glycine to an arginine at position 299 alters the charge distribution on the protein surface. One or both of these changes may contribute to the susceptibility of individuals to develop inflammatory bowel diseases and asthma through one of the following molecular mechanisms: (i) The difference in the inherent stability of the GSDMB variants and/or their susceptibility to cellular protein degradation machinery may lead to different cellular concentrations, which in turn may affect disease risk; (ii) different loop conformations and surface charges of the two GSDMB variants may lead to altered interactions between the N- and C-terminal domains, thus affecting protein activity; and (iii) the two GSDMB variants may interact differently with protein partners, such as the reported HSP90β and fatty acid synthetase (8).

GSDMB Stability and Caspase Cleavage Profile.

To investigate these possible mechanisms, circular dichroism was used to measure the thermal stability of the GSDMB_C variants (encompassing amino acids Asp244–Ser411). The CD spectra of both GSDMB_C Arg299:Ser306 and Gly299:Pro306 variants show no thermal unfolding transition upon heating from 22 °C to 90 °C. This result reveals a highly stable C-terminal domain and indicates that the polymorphism residues at positions 299 and 306 do not affect protein stability at physiological temperature. Thus, GSDMB stability is not a likely mechanism underlying the complex-trait inflammatory disease risk.

Shi et al. have shown that GSDMB is not a substrate for the inflammatory caspases 1 and 4/5/11 whereas GSDMD is cleaved by these caspases in the interdomain linker (29). We investigated whether GSDMB is a substrate of the human apoptotic caspases and found that His₆-GSDMB, MBP-GSDMB, and MBP-GSDMB_N are substrates of the apoptotic executioner caspases 3, 6, and 7 (Fig. 5). The higher molecular weight cleavage products from His₆-GSDMB and MBP-GSDMB_N are recognized by the anti-GSDMB_C and anti-MBP antibodies, respectively (Fig. 5, Bottom). The molecular weights of the proteolytic fragments indicate that the caspase recognition site is in the N-terminal domain of GSDMB and not in the interdomain linker region. N-terminal sequencing of the C-terminal fragment from a caspase-3 cleavage reaction revealed amino acids ₉₂STGELIVRLP₁₀₁ (Fig. 1), and thus the cleavage site is after ₈₈DNVD₉₁. Interestingly, the CaspDB lists this site with the highest caspase cleavage probability (39). Thus, human apoptotic executioner caspases did not generate an entire GSDMB N-terminal domain, analogous to the pyroptotic N-terminal domain of GSDMD.

The loop containing the polymorphism amino acids also contains another predicted caspase recognition site, ₃₀₄EDPD₃₀₇, albeit with a probability score of 0.803. All four MBP-GSDMB_C variants (Gly299:Pro306, Arg299:Ser306, Gly299:Ser306, and Arg299:Pro306) were digested with caspases 1 to 3 and 6 to 10, but none was cleaved. The functional significance of caspase 3/6/7 cleavage at an internal GSDMB’s N-terminal domain site and its impact on the protein activity are currently unknown.

"VSports在线直播" GSDMB_N Binds Phosphoinositides and Sulfatide.

Ding et al. first discovered that the N-terminal domains of Gsdma3, GSDMA, and GSDMD bind phospholipids (25). The C-terminal domains inhibit phospholipid binding to the full-length proteins, but not to noncovalent N+C complexes. The identified phospholipids were components of the cell membrane’s inner leaflets, including monophosphoinositides [PtdIns(3)P, PtdIns(4)P, and PtdIns(5)P], bisphosphoinositides [PtdIns(3,4)P₂, PtdIns(3,5)P₂, and PtdIns(4,5)P₂], triphosphoinositide [PtdIns(3,4,5)P₃], and cardiolipin [1,3-bis(sn-3′-phosphatidyl)-sn-glycerol]. Subsequent reports confirmed the binding of GSDMD_N to PtdIns(4)P, PtdIns(3,4)P₂, phosphatidic acid, and cardiolipin, and weakly to phosphatidylserine (32). Because cardiolipin is a component of the inner leaflet of mitochondrial membrane, the binding data substantiated previous results that Gsdma3 is transported into the mitochondria to disrupt mitochondrial membranes (26, 27) although mitochondrial localization was not reported for GSDMD. For GSDMD, phospholipid binding enables higher order oligomerization of the GSDMD N-terminal domain in the lipid bilayer and formation of ring structure-like pores to disrupt membrane structure. These data also imply either that the interfaces between the two domains of intact Gsdma3, GSDMA, and GSDMD overlap with the phospholipid binding sites on the respective N-terminal domains or that the GSDMs’ C-terminal domains induce protein conformations in the vicinity of the binding sites that impede lipid binding.

We screened the lipid binding activity of MBP-GSDMB_N, MBP-GSDMB_C, full-length MBP-GSDMB, and His₆-GSDMB using the nitrocellulose membrane immobilized with phosphoinositides, membrane lipids, and sphingolipids. The GSDMBs containing the N-terminal domain bound mono-, bis-, and triphosphoinositides (Fig. 6). Weaker binding activities were also observed for phosphatidic acid, phosphatidylglycerol, sulfatide (3-O-sulfogalctosylceramide), and cardiolipin. In contrast, all four MBP-GSDMB_C variants and His₆-MBP control did not bind any lipids. Thus, the lipid-binding activity of GSDMB is also located in its N-terminal domain. Additional experiments with the lipid arrays, nitrocellulose membrane spotted with varied lipid amounts, confirmed most but not all of the initial binding activities of GSDMB and highlighted variability in the lipid-binding affinities of the full-length and N-terminal domain of GSDMB (Fig. 6B). Strikingly, unlike other gasdermins, the presence of the GSDMB C-terminal domain in the full-length protein did not prevent phospholipid binding. This finding and the affinity for sulfatide, not for cardiolipin, distinguish GSDMB from Gsdam3/GSDMA and GSDMD. The lipid-binding activity of intact GSDMB could be due to either that (i) the interdomain interaction between the N- and C-terminal domains of GSDMB is weaker than that of the other gasdermins or (ii) that the phospholipid binding site on the GSDMB N-terminal domain is not in the vicinity of the interdomain interface, as suggested for GSDMA (25). Currently there is no evidence that a stable N-terminal domain is generated from a proteolytic cleavage of GSDMB. Thus, unlike GSDMA/Gsdma3 and GSDMD, a pyroptotic activity of the recombinant GSDMB N-terminal domain, as reported by Ding et al. (25), may be decoupled from its phospholipid-binding activity because the full-length GSDMB is noncytotoxic despite similar lipid binding properties.

Liposome-binding experiments were performed to confirm that the unique sulfatide binding to GSDMB_N is not an artifact due to unspecific charge–charge interactions. These studies showed that, indeed, the N-terminal domain bound liposomes containing sulfatide whereas the C-terminal domain did not.

"VSports" GSDMB May Have a Different Interdomain Interaction than That of Gsdma3.

To gain insights into the lipid-binding property of the full-length GSDMB, we compared the structures of the GSDMB_C and Gsdma3 (25). The topology of the shared seven-helix bundle is conserved, and the rmsd of 113 Cα positions shared between the C-terminal domains of GSDMB and Gsdma3 is 2.3 Å (Fig. 7). However, examination of the superposed structures suggests that there are important differences that may lead to a different interdomain orientation of GSDMB compared with Gsdma3 (and perhaps also GSDMA and GSDMD), which may explain why the C-terminal domain of GSDMB does not inhibit lipid binding. First, the conformations of the GSDMB loop containing the polymorphism residues and that of Gsdma3 are entirely different (colored red in Fig. 7). Second, another region within the GSDMB and Gsdma3 C-terminal domains is strikingly different (colored blue in Fig. 7). The GSDMB_C flexible loop (Met366–Tyr382) that connects the last two helices (α10–α11) has a counterpart segment in Gsdma3, which is 33 aa longer (Pro384–Asp433, colored blue in Fig. 7). The Gsdma3 Pro384–Asp433 segment links α10 and α12 helices and forms a subdomain comprising an α-helix (α11) and a 3 β-stranded sheet (β12–β14) (Fig.1). Notably, helix α11 of Gsdma3 extends the number of helices of its C-terminal helix bundle to eight. This Gsdma3 subdomain interacts with Gsdma3 α4 (Pro179–Asn187) within a largely disordered N-terminal domain loop (Thr168–Asn193) that has been shown by site-directed mutagenesis (replacement of Gsdma3 Leu184 or GSDMD Leu192 with an Asp) to be involved in membrane disruption (25). For the shorter GSDMB loop (Met366–Tyr382) to regulate the interaction of the N-terminal domain with cellular membranes, the interdomain orientation must change.

Third, three Gsdma3 N-terminal domain segments interact with the C-terminal domain (colored magenta in Fig. 7): the N-terminal α1 helix (Met1–Asn16), an Ω-loop connecting β1 and β2 (Lys42–Arg52), and the structurally defined α4 (Pro179–Gln187) of the otherwise disordered membrane-disrupting loop (Thr168–Asn193). By comparison, the predicted Ω- and membrane-disrupting loops in the N-terminal domain of GSDMB are shorter by 3 and 4 aa (Fig.1), and they cannot adopt the same conformations as those observed in the Gsdma3 structure. To accommodate all these changes, the mode of interactions between the N- and C-terminal domains of GSDMB and Gsdma3 are likely to be different.

Finally, of the six alternative splice variants of the GSDMB, five have interdomain linkers that vary in length and sequence. These variations are likely to modulate the mode of interdomain interactions and perhaps also the GSDMB affinities to lipids and protein partners. Indeed, the 394-aa-long GSDMB splice variant (NCBI NP_061000.2, GSDMB-2) exhibited different activities than the 403-aa splice variant (NCBI NP_001035936.1, GSDMB-1). Overexpression of the GSDMB-2 variant led to increased activation of Rac-1 and Cdc-42 and metastatic burden compared with overexpression of GSDMB-1. In addition, although both GSDMB splice variants interacted with HSP90, only GSDMB-2 relied on the chaperone for its stability (8). We speculate that the longer or shorter linker segments, as well as the different linker amino acid sequences of splice variants, may modulate protein activity and/or direct the protein to different subcellular compartments and cell fates.

"V体育ios版" Implications for GSDMB’s Function in Health and Disease.

The structures reported here reveal the conformational and surface electrostatic differences that accompany the GSDMB_C amino acid replacements encoded by SNPs linked to increased risk of developing complex-trait inflammatory diseases, such as asthma, Crohn’s, and ulcerative colitis. The GSDMB C-terminal domain does not inhibit the lipid-binding activity of its N-terminal domain, as reported for other gasdermins. Therefore, amino acid replacements in the GSDMB C-terminal domain offer a nuanced mechanism to regulate protein activity. It is currently unknown whether the C-terminal domain modulates the lipid selectivity and/or binding affinity by directly interacting with the N-terminal domain or by associating with a partner protein such as HSP90β or fatty acid synthetase. In addition to phosphoinositides, the binding of sulfatide to GSDMB seems to be unique and was not observed for GSDMA, Gsdma3, and GSDMD (25, 32). Sulfatide is synthesized in the Golgi apparatus from galactosylceramide and then is distributed to the extracellular leaflet of myelin, to the apical membrane of epithelial cells, and also to the lysosome for degradation. Lipodomic analysis showed a dramatic increase in sulfatide level, with progression of epithelium polarization, a process that protects the integrity of the epithelial cell barrier (40). Elevated levels of sulfatide are documented in many cancers. Abundant sulfatide on the surface of cancer cells may serve as a native ligand for P-selectin, a protein that promotes cell migration and metastasis (41). Sulfatide also stimulated phosphorylation of transcription factor Sp1 in hepatocellular carcinoma cells through Erk1/2 signaling, which in turn increased expression of the integrin αV subunit, another protein involved in metastasis (42). In ulcerative colitis, the mucosa is enriched with type II NKT cells, and sulfatide binds to these cells to induce production of IL-13, which mediates epithelial cell cytotoxicity (43). In asthma, transgenic mice overexpressing the human GSDMB developed asthmatic symptoms, and an overexpression of GSDMB in bronchial epithelial cells induced the expression of 5-lipoxygenase, which in turn induced TGF-β1 expression (44). 5-Lipoxygenase converts arachidonic acid and the omega-3 fatty acid icosapentaenoic acid into the signaling lipids leukotrienes, and sulfatide inhibits the enzyme (45). Currently, cytoplasmic protein(s) that specifically transport sulfatide between cellular compartments have not been identified. Could the GSDMB be a part of the epithelial cell transport system that targets sulfatide to the plasma membrane? Then, overexpression of GSDMB might be responsible for higher sulfatide levels on the apical surface of epithelial cells. For the GSDMB SNPs associated with inflammatory bowel diseases and asthma, if expression of the GSDMB R299:S306 SNP variant protein leads to aberrant sulfatide transport, this perturbation might compromise the integrity of the epithelial cell barrier and/or promote inflammatory processes.

In conclusion, we report molecular properties that distinguish GSDMB from other GSDMs, providing evidence to explain the different biology associated with the oncogenic GSDMB and the apoptotic GSDMA, GSDMC, and GSDMD. First, intact, full-length GSDMB binds signaling lipids in contrast to other GSDMs, whose C-terminal domains inhibit lipid binding, and only their free N-terminal domains can bind lipids, form pores, and kill cells. Second, we discovered that GSDMB binds sulfatide, but not cardiolipin, in contrast to other GSDMs, which exhibit the reverse binding specificity. The reported involvement of sulfatide on the surface membrane of epithelial cells in some cancers and complex trait inflammatory disease suggests that GSDMB may function in sulfatide cellular transport. In addition, GSDMB and other GSDMs also bind phosphoinositides. The biological implications of these phosphoinositide-binding activities are currently unknown and warrant further investigations. Third, the structures of the GSDMB C-terminal domain show that the SNPs associated with complex trait inflammatory disease alter the flexibility and charge of a surface loop, properties that might be responsible for activity modulation. It remains to be discovered whether the altered loop properties exert an intramolecular effect (i.e., via an interaction with GSDMB’s N-terminal domain) or an intermolecular effect (i.e., via an unknown protein–protein interaction). Nevertheless, the structure analysis offers a potential disease mechanism in support of GWAS data that implicated GSDMB polymorphism in disease risk.

VSports在线直播 - Cloning, Expression, and Protein Purification.

The human GSDMB gene was amplified from a cDNA clone (ID 5205060; Open Biosystems, Inc.) and was ligated into the NotI and NheI sites in the pMALX(E) vector (kindly provided by Lars C. Pedersen, National Institute of Environmental Health Sciences, Research Triangle Park, NC) for expression in E. coli. The maltose-binding protein (MBP) contains five mutations, D82A/K83A/E172A/N173A/K239A, and a 3-alanine extension at the C terminus. Point mutations in GSDMB_C were introduced using the QuikChange mutagenesis protocol (Stratagene) and were confirmed by sequencing. The recombinant MBP-GSDMB_C protein spans Lys1 to Ala370 of MBP and Met220 to Ser411 of GSDMB_C. E. coli BL21*(DE3) cells transformed with plasmid were grown in LB medium containing 0.1 mg/mL ampicillin for 16 h at 37 °C. The culture was diluted 1:100 into fresh LB-Amp medium, and the cells were grown at 37 °C to a density of OD₆₀₀ ∼0.6. The incubation temperature was reduced to 22 °C before the addition of 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and the cells were grown for 16 h. Cells transformed with the MBP-GSDMB_N gene (MBP fused to GSDMB Phe2-Ilu226) were grown at 22 °C to a density of OD₆₀₀ ∼0.2, before the addition of 1 mM IPTG, and were grown for 16 h at 15 °C. For the MBP-GSDMB_N and MBP-GSDMB_C fusion proteins, cells resuspended in 50 mM Tris⋅HCl (pH 8.0), 0.1 M NaCl, 1 mM ethylene-diamine-tetra-acetic acid (EDTA), 10% (vol/vol) glycerol, 0.17 mg/mL lysozyme, and 3.3 units/mL benzonase (Novagen) were lysed by sonication. The cell lysate was centrifuged for 1 h at 23,700 × g at 4 °C, and the soluble fraction was applied onto an amylose column (NEB), equilibrated in 50 mM Tris⋅HCl (pH 8.0), 0.10 M NaCl, 0.5 mM EDTA. After washing the column, the bound protein was eluted with the above buffer containing 10 mM maltose. The protein was further purified using size exclusion chromatography with a Sephacryl S200 column equilibrated in 50 mM Hepes (pH 7.3), 0.1 M NaCl, 0.02% NaN₃, 10 mM maltose at 4 °C. Full-length MBP-GSDMB (MBP fused to GSDMB Phe2-Ser411) was coexpressed in the presence of chaperone proteins, GroEL/GroES, following the manufacturer protocol (TAKARA BIO Inc.). E. coli BL2*(DE3) cells transformed with expression vectors for MBP-GSDMB and GroEL/GroES were grown at 37 °C in the presence of 50 μg/mL ampicillin, 20 μg/mL chloramphenicol, and 0.5 mg/mL l-arabinose to a density of OD₆₀₀ ∼0.8, before the addition of 0.2 mM IPTG, and then grown for 4 h. MBP-GSDMB was copurified from an Amylose affinity column with GroEL/GroES. The chaperones were removed by precipitation of MBP-GSDMB with 1.8 M ammonium sulfate. MBP-GSDMB was further purified by a Superdex 200 size exclusion column. For the His₆-GSDMB protein, GSDMB was cloned into the baculovirus transfer vector, pVL1393 (BD Biosciences), with an N-terminal His₆-tag and seven-residue tobacco etch virus protease cleavage site. The engineered pVL1393 was cotransfected with Sapphire Baculovirus DNA (Allele Biotechnology) into Sf9 cells (Gibco). The recombinant baculovirus was used to transfect Sf9 cells grown in SF-900 II SFM (Gibco) at 27 °C. Cells were harvested 4 d posttransfection and lysed in 150 mL of 10 mM Tris⋅HCl (pH 7.5), 0.15 M NaCl, 1% (vol/vol) Nonidet P-40, 1 mM DTT, and protease inhibitor mixture. The lysate was centrifuged at 212,795 × g for 1 h, and the supernatant was applied to an Ni²⁺-NTA column (Qiagen). His₆-GSDMB was eluted with the above buffer containing 10 to 250 mM imidazole and further purified using a phenyl Sepharose column. The protein concentrations were determined at 280 nm wavelength with a calculated extinction coefficient of 74,955 M⁻¹⋅cm⁻¹, for MBP-GSDMB_C, 86,415 M⁻¹⋅cm⁻¹ for MBP-GSDMB_N, 95,020 M⁻¹⋅cm⁻¹ for MBP-GSDMB, and 30,160 M⁻¹⋅cm⁻¹ for His₆-GSDMB.

VSports app下载 - Crystallization, Data Collection, and Structure Determination.

Crystals of MBP-GSDMB_C Gly299:Pro306 were obtained at room temperature using the vapor diffusion method with 3-μL drops comprising equal volumes of protein (containing 10 mM maltose) and reservoir solution containing 2.1 M sodium malonate (pH 6). The MBP-GSDMB_C Arg299:Ser306 variant and Gly299:Ser306 variant crystals were obtained using reservoir solutions of 2.2 M sodium malonate (pH 6) and 9 mM hexamine CoCl₃ or 10 mM EDTA, respectively. For data collection, crystals were transferred to 2.6 M sodium malonate (pH 6) and 10 mM maltose, supplemented with either 9 mM hexamine CoCl₃ or 10 mM EDTA, and flash-cooled in liquid nitrogen. Diffraction data were collected at the General Medicine and Cancer Institute (GM/CA, 23ID-B) and the Structural Biology Center (SBC, 19ID) Collaborative Access Team beamlines at the Advanced Photon Source (Argonne National Laboratory). Diffraction data were processed with MOSFLM (46). The structure was determined by molecular replacement using the program PHASER (47) with the MBP structure (PDB ID code 3H4Z) as the search model for the MBP-GSDMB_C Arg299:Ser306 variant structure. The MBP-GSDMB_C Arg299:Ser306 structure then served as the search model for the two additional structures. The phase improvement program PARROT (48) was used to improve the initial electron density maps. Structure refinement and model building were performed with the program REFMAC5 (49) and the interactive computer graphics program COOT (50). Figures were prepared with PyMOL (The PyMOL Molecular Graphics System; Schrödinger, LLC) and ESPript 3 (51).

Protein Lipid Overlay Assay. (VSports最新版本)

Nitrocellulose strips and arrays of phosphoinositides (PIPs), membrane lipids, sphingophospholipids (Echelon Biosciences), and nitrocellulose arrays of cardiolipin and sulfatide (prepared as described) (52) were blocked in Tris-buffered saline containing 0.1% (vol/vol) Tween 20 and 3% fatty acid-free BSA (TBST-BSA; Sigma) for 1 h at room temperature. The membranes were incubated with 2 to 3 μg/mL MBP-GSDMB, MBP-GSDMB_N, MBP-GSDMB_C, or His₆-MBP in TBST-BSA buffer overnight at room temperature. After three washes with TBST-BSA, the membranes were blotted with anti-MBP monoclonal antibody conjugated to HRP, or with anti-GSDMB C-terminal monoclonal antibody and goat anti-mouse IgG HRP conjugate. The blots were visualized using Supersignal West Pico enhanced chemiluminescence substrate.

For liposome-binding assays, liposomes containing 4:1 molar ratio egg l-α-phosphatidylcholine and porcine brain sulfatide (Avanti Polar Lipids) were prepared as previously described (53). MBP-GSDMB_N, MBP_GSDMB_C or GSDMB_C were incubated with 0.5 to 1 mM liposomes for 30 min at 22 °C in an 80-μL reaction volume. The samples were centrifuged in a Beckman TL-100 μL tracentrifuge for 20 min at 4 °C at 128,000 × g. The supernatant was collected. The pellet was washed with 100 μL of buffer [100 mM NaCl, 20 mM Hepes/NaOH (pH 7.5), and 0.02% sodium azide] and recentrifuged, and buffer was added to obtain the same volume as that of the supernatant. The proteins in the supernatant and pellet fractions were analyzed by SDS/PAGE and Coomassie blue staining.

In Vitro Caspase Cleavage Assay.

Active human recombinant caspases 1, 2, 3, 6, 7, 8, 9, and 10 were purchased from BioVision. Approximately 1 µM His₆-GSDMB, or 3.5 µM MBP-GSDMB, or 1.6 µM MBP-GSDMB_N, or 1.3 µM MBP-GSDMB_C was incubated with 1 unit of active caspases at 37 °C for 2 h in a 25-µL buffer [50 mM Hepes (pH 7.3), 50 mM NaCl, 10 mM EDTA, 0.1% CHAPS, 10 mM DTT, 5% glycerol]. The cleavage products were resolved by 14% (wt/vol) SDS/PAGE under denaturing conditions, and Western analyses were conducted as described above. For the mapping of the caspase cleavage site on GSDMB, a pET21b vector containing the Caspase 3 gene (kindly provided by A. Clay Clark, University of Texas at Arlington, Arlington, TX) was purified as previously described (54).