Fig. 4
From: Structural and functional insights into the lipopolysaccharide ABC transporter LptB2FG
LptB2FG cavity and the periplasmic domains. The color scheme is the same as in Fig. 2. a The transmembrane domains of LptB2FG, top view. Both LptF and LptG have six TM segments respectively, forming a cavity. b The superimposition of LptF and LptG from K. pneumoniae. The proline substitutions are shown in sphere. c Functional assays of the single proline mutants of periplasmic domains of LptF and LptG according to E. coli amino-acid sequences. NR1113 cells were transformed with empty vector (pTRC99a_Kan, negative control) or the vector encoding LptBF(Flag)G(Myc) (positive control) or its single proline mutant derivatives. All bacterial cultures were adjusted to an OD600 nm = 0.5 with fresh medium, serially dilution 1:10 as indicated on the top of the figure and then replica plated in agar plates containing kanamycin but in the absence of L-arabinose. The positive control (lptBF(Flag)G(Myc) in NR1113), the negative control (empty plasmid in NR1113), LptF mutants F_D229P, F_Q231P, F_R223P and F_T225P, and LptG mutants G_S223P, G_T225P, G_G228P, and G_W230P were used in the functional assays. Double proline substitutions (F_D229P/Q231P, F_T225P/R223P, G_G228P/W230P, and G_T225P/S223P) are reported in Supplementary Fig. 2. d The protein expression levels of the mutants shown by the western blotting. The LptF and LptG protein expression of the negative control (empty plasmid), the positive control (plasmid with LptBF(Flag)G(Myc)), LptF mutants D229P, F_Q231P, F_R223P, and F_T225P, and LptG mutants G_S223P, G_T225P, G_G228P, and G_W230P were detected. The bacteria cells for western blotting were cultured in the presence of 0.2% L-arabinose. All mutants in Figs. 3 and 4 and Supplementary Fig. 2 are based on E. coli amino-acid sequence. The difference in residues is listed here: LptF-D229 (N229_K. pneumoniae), LptG-T225 (M225_K. pneumoniae)
