recombinant s cerevisiae ulp1  (Addgene inc)

Journal: bioRxiv

Article Title: NLRP3 acts as a direct sensor of intracellular potassium ions

doi: 10.64898/2026.03.12.707678

Figure Lengend Snippet: (A) Immunoblot of purified full-length NLRP3 (3-1036) in K + (150 mM KCl) or Na + (150 mM NaCl) buffer, with ± MCC950 added prior to pronase digestion. Anti-NLRP3 antibodies were used. Data are representative of n=5 independent experiments. B) Quantification of A showing combined data from n=5 independent experiments. One-way ANOVA with Dunnett’s multiple comparison test. (C-E) Melting temperature analysis (Tm, °C) of recombinant human FISNA-NACHT NLRP3 (131–679) or ULP1 in buffers containing increasing KCl and decreasing NaCl concentrations (total ion concentration 150 mM) ± MCC950 (C), or ± ADP (D) or using RbCl instead of KCl (E). Data show n=3 technical replicates (mean ±SD). One sample t-test. (F) As in A but with purified human FISNA-NACHT NLRP3 (131–679) protein. Data are representative of n=5 independent experiments. (G) Quantification of F combining n=5 independent experiments. One sample t-test. (H) Mass spectrometric peptide fingerprint of full-length NLRP3 immunoprecipitated with the specific NLRP3 antibody D4D8T from NLRP3-Luc-expressing HEK293T cells and purified after SDS-PAGE (Fig. S2I). Data from 1 independent experiment. (I) Representative snapshot from µs-scale MDS showing K⁺ ion density aggregated over time (magenta) within the nucleotide binding pocket of the NLRP3 NACHT domain and in proximity to Thr233. Trajectories and densities accumulated from n=3 independent MDS. (J) Time-resolved occupancy analysis of K⁺ ions within 5 Å sphere of individual NLRP3 residues (blue ID: residue is part of the NBD-HD1/WHD-HD2 interface; cyan ID: residue is within a 5 Å sphere of ADP, i.e. within nucleotide binding pocket; orange ID: residue is part of one of the NACHT domain surface loop region) across n=3 independent MDS (A-C) of the FISNA-NACHT structure, depicted in a heat map showing only residues with > 10% accumulated contact frequency (green = 1 K⁺ ion, black = ≥2 K⁺ ions; color code indicates to subdomains and grayscale heatmap indicates % occupancy averaged across all shown MDS). The graph depicts % occupancy for all FISNA-NACHT residues. (K) Comparison of NACHT subdomain orientations (NBD-HD1 part in white, WHD-HD2 in grey) in inactive cage structure (left) vs active disk (right). The distance between Thr233 and His522, both nucleotide binding pocket and K + interacting residues, is taken as a proxy to illustrate the shift from inactive to active conformation.

Article Snippet: In brief, recombinant NLRP3 proteins described above were diluted to 0.5 mg/mL in buffers containing 20 mM Tris (pH 7.5), 5 mM MgCl 2 , and various KCl and NaCl concentrations as indicated in the graphs (with a 150 mM total of salt concentration) in presence or absence of 800 μM MCC950 and incubated on ice for 15 min. Recombinant S. cerevisiae ULP1 (Addgene #64697, a gift from Hideo Iwai, Institute of Biotechnology, Helsinki, Finland) was purified from E. coli and used as a negative control ( ).

Techniques: Western Blot, Purification, Comparison, Recombinant, Concentration Assay, Immunoprecipitation, Expressing, SDS Page, Binding Assay, Residue