Fig. 3.

The transmembrane domains of LptB₂FG and the lateral openings. The color scheme is the same as in Fig. 2. a The lateral gate of TM1G-5F interaction. The residues K40 of TM1G and S318 of TM5F forming the contact are shown in sphere and stick, respectively. The positively charged and highly conserved residues K34 and R136 of LptG are also shown in sphere. b The lateral gate of TM1F-5G interaction. There are five interactions within TM1F-5G (F_V32/G_I325, F_V32/G_Q324, F_V39/G_L331, F_L35/G_P328, and F_I25/G_ F317), shown in stick. The hydrophobic residues F26 and L62 of LptF are shown in sphere. The TM1F(G) crosses the IM at an angle of 67° and 53° to the membrane plane, respectively. c Functional assays of the double mutants LptG K34E/R136E, LptG K40E/K41E, and the LptF F26D/L62D in NR1113 depletion strain. NR1113 cells were transformed with the empty vector (pTRC99a_Kan, negative control) or the vector encoding LptBF(Flag)G(Myc) (positive control) or its mutant derivatives. All bacterial cultures were adjusted to an OD₆₀₀ = 0.5 with fresh medium, serially diluted 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. Row 1: NR1113 cells transformed with LptBF(Flag)G(Myc) plasmid was used as a positive control. Row 2: NR1113 cells transformed with the empty plasmid (pTRC99a_Kan) was used as the negative control. Row 3: LptG double-mutant K34E/R136E. Row 4: LptG double-mutant K40E/K41E. Row 5: LptF double-mutant F26D/L62D. d Detection of protein expression levels of LptF(Flag)G(Myc) of the negative control, positive control, and the mutants of G_K34E/R136E, G_K40E/K41E, and F_F26D/L62D by western blotting. The bacterial cells for western blotting were cultured in the presence of 0.2% L-arabinose