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URL : https://hal.archives-ouvertes.fr/pasteur-02554309

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. Contact-for and . Sharing, Contact should be directed to Pedro Alzari at pedro.alzari@pasteur.fr METHOD DETAILS Protein Expression, Purification and Kinase Assay His-tagged recombinant CpxR RD , CpxR RD_D51N and CpxA HDC were expressed in E. coli BLI5 cells and purified by immobilised metal affinity chromatography followed by gel filtration using standard procedures. Purified proteins were concentrated by ultrafiltration (Vivaspin, Sartorius), frozen in liquid nitrogen and stored at -80 C before use. The CpxR RD_D51N mutant was constructed by sitedirected mutagenesis using a

. Mechaly, Membranes were isolated, solubilized in 1.5% (w/v) n-dodecyl-b-D-maltoside and applied onto an Ni-NTA column (1 ml) equilibrated with 50 mM Tris-HCl pH 8.0, 0.1% Brij35, 300 mM NaCl, 5% glycerol, and 20 mM imidazole. After extensive washes with the same buffer, the protein was then eluted with a 20-300 mM imidazole gradient. CpxA-containing fractions were pooled and dialyzed against 25 mM Hepes pH 7.8, 100 mM NaCl, 50 mM KCl, 10 % glycerol, and 0.05% Brij35. Autokinase activity on full-length CpxA (as Brij35 complexes) were performed using, 2011.

X. , Crystallography Crystals were grown using the vapor diffusion method at 18 C. Drops were set by mixing equal volumes of protein and reservoir solutions. Prior to crystallization drops setup, CpxA HDC (30 mg/ml) was incubated with 3 mM ATP and 1 mM MgCl 2 for 2-3 minutes at room temperature. Phosphorylated CpxA HDC crystals were grown in 100 mM Tris (pH 8.5), 1.5 M ammonium sulphate and 12 % v/v glycerol. Plate-shaped crystals of CpxR RD appeared within two weeks in wells containing 100 mM HEPES

. Lima, CpxR RD crystals were soaked with 50 mM acetyl-phosphate (AcP), a proven substrate for CpxR, 2012.

(. Rrs and . Klein, All crystals were flash-frozen and stored in liquid nitrogen before data collection under cryogenic conditions (100 K)

. Mechaly, The crystal structure was solved by molecular replacement (MR) using the program Phaser (McCoy et al., 2007) and a previously determined CpxA HDC structure (PDB entry 4BIW) as search probe, Diffraction data were collected at synchrotron beamlines Proxima 1 (Soleil), ID23-1 and ID29 (ESRF), 2013.