Preparation of recombinant FixL and FixJ proteins

B. japonicum FixL and FixJ genes encoding full-length FixL and FixJ and truncated proteins [FixL_PAS-PAS (residues 1 to 275) and FixJ_N (residues 1 to 124)] were separately amplified by polymerase chain reaction (PCR) with PfuTurbo DNA polymerase (Agilent Technologies). The PCR fragments were cleaved by Bsr GI and Avr II for FixL, Bsr GI and Nhe I for FixJ (New England Biolabs), and cloned into 5′Bsr GI–3′Avr II sites of pET-47b(+) vector (Novagen) for expression with hexa-His tag, followed by the HRV3C protease cleavage site in the N terminus of those proteins. Site-directed mutagenesis of the coiled-coil region in full-length FixL was performed by QuikChange protocol (41) using pET-47b(+) vector inserted in the full-length FixL gene as a template.

Escherichia coli BL21(DE3) cells (Nippon Gene) carrying these plasmids were inoculated in terrific broth (TB) containing kanamycin (50 μg/ml; Wako) and 1% glucose for 4 hours at 37°C with shaking at 150 rpm. One milliliter of the preculture solution was inoculated into 300 ml of TB medium containing kanamycin [50 μg/ml; plus 250 μM 5-aminolevulinic acid (Cosmo Energy Holdings) only for the expression of FixL]. The cultivation was done at 37°C with shaking at 120 rpm. After 4 hours of cultivation, expression of FixL or FixJ was induced with 0.3 or 0.2 mM isopropyl-β-d-thiogalactopyranoside, respectively, and cultivation was allowed to continue for another 15 hours at 23°C with shaking at 80 rpm. The cells were harvested by centrifugation at 4000g for 10 min, and the cells were washed in 30 mM tris-HCl (pH 8.0) twice.

Purification of FixL and FixJ was performed by the following same steps at 4°C. Harvested cells were resuspended in a lysis buffer [50 mM tris-HCl (pH 8.0), 300 mM NaCl, 10% (w/v) glycerol, and one tablet of cOmplete EDTA-free protease inhibitor cocktail (Roche)]. The lysate was mixed with lysozyme (0.1 mg/ml; Sigma-Aldrich), deoxyribonuclease I (0.05 mg/ml; Sigma-Aldrich), and 5 mM MgCl₂ for 30 min and disrupted by Microfluidizer M-110Y (Microfluidics). Cell debris was removed by ultracentrifugation at 40 krpm for 1 hour. The supernatant was loaded onto a HisTrap HP (GE Healthcare) column equilibrated with buffer A [50 mM tris-HCl (pH 8.0), 300 mM NaCl, 10% (w/v) glycerol, 10 mM imidazole/HCl (pH 8.0)]. FixL or FixJ was eluted by an imidazole concentration gradient (0 to 300 mM). The eluted fractions were treated with N-terminal 6×His-tagged HRV3C protease (produced in-house) to remove the 6×His-tag from the recombinant FixL and FixJ proteins, and the solution was dialyzed with buffer B [40 mM tris-HCl (pH 8.0), 150 mM NaCl, 10% (w/v) glycerol]. After the dialysis, the protein solution was loaded to HisTrap FF (GE healthcare) equilibrated with buffer B to remove the HRV3C protease and remaining His-tagged proteins, and the flowthrough was collected. The collected solution was concentrated by Amicon Ultra-15 (Merck Millipore) and centrifuged at 15 krpm for 20 min. The supernatant was loaded to HiLoad 16/600 Superdex 200 (GE healthcare) equilibrated with buffer B. The purity of FixL or FixJ was checked by SDS–polyacrylamide gel electrophoresis (PAGE). The purified samples were mixed with 2× SDS-PAGE buffer containing 125 μM tris-HCl (pH 6.8), 4% SDS, 20% (w/v) sucrose, 0.01% (w/v) bromophenol blue (BPB), and 10% (v/v) 2-mercaptoethanol and boiled at 95°C for 10 min before the electrophoresis. NuPAGE Bis-Tris gels (10%; Thermo Fisher Scientific) were used for the electrophoresis with NuPAGE Mops SDS running buffer (Thermo Fisher Scientific) for FixL and NuPAGE MES SDS running buffer (Thermo Fisher Scientific) for FixJ. The gels were stained by EzStain AQua (ATTO). In FixL, the highly purified fractions with an R_z (A_398nm/A_280nm) value of >1.3 were used for SAXS studies. These spectra were measured in 40 mM tris-HCl (pH 8.0), 150 mM NaCl, and 10% (w/v) glycerol at 20°C by NanoDrop 2000c spectrophotometers (Thermo Fisher Scientific). Because His-tagged full-length FixL showed the kinase inhibition upon cyanide binding to the heme and nearly the same phosphotransfer activity as the His-tag–removed protein, we used the His-tagged full-length FixL proteins in the phosphorylation activity assays of the coiled-coil mutants (fig. S9).