Skip to main content
. 2020 Aug 3;3:418. doi: 10.1038/s42003-020-01147-1

Fig. 4. Structural and biochemical analysis of the lipid-binding pocket in MucB.

Fig. 4

a A polyethylene glycol (PEG) molecular bound in the hydrophobic cavity of MucB structure. The hydrophobic residues surrounding PEG are shown with green sticks. The polyethylene glycol monomethyl ether 550 is shown in hot-pink stick and its 2mFo-DFc map (1.5σ) is displayed as blue mesh. b SDS-PAGE assay of MucAperi (125 μM) degradation by AlgW (25 μM) in the presences of MucB (130 μM), YVF peptide (80 μM), and different reagent including 0.1 mM lipid-A hydrolyzed with NaOH (L-IIA was colored in red), 0.1 mM LPS (Solarbio) or boiled LPS, n-dodecyl-β-d-maltopyranoside (0.05% DDM), n-nonyl-β-d-glucopyranoside (0.05% NG), n-octyl-β-d-glucopyranoside (0.05% β-OG), polyethylene glycol monomethyl ether reagents like 1% PEG550, 1% PEG2000, 1% PEG3350, 1% PEG5000, (+/−)-2-methyl-2,4-pentanediol (1% MPD), 1% DMSO, 1% isopropanol, 1% glycerol, 15 mM disaccharide (maltose, β-d-glucopyranosyl-d-glucose) or 1 mM different chain length fatty acids (Caprylic acid (C8), Decanoic acid (C10), Lauric acid (C12), Myristic acid (C14), Palmitic acid (C16)). The fractions were incubated in degradation buffer (25 mM Tris–HCl, PH 7.5, 150 mM NaCl) at 37 °C for 30 min. c ELISA assay characterizing the interactions of lipid-A with MucAperi, MucB, MucB-NTD and mutants. lipid-A was coated in Nunc-Immuno™ MicroWell™ 96-Well Plates at a final amount of 50 μM/well. And then twofold serial dilutions of each indicated his-tag proteins were prepared. The concentration gradient is range from 100 to 0.05 μM. Following capture by lipids, the MucAperi, MucB and MucB mutants were incubated by His-Tag antibody and horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody. Finally, the bound proteins were detected using TMB-ELISA substrate solution and quantified at 450 nm. Each experiment was performed three times, and each point is a mean of three replicates ± SD. d The sensitivity of MucB to DDM. MucB and mutants (concentration were both 130 μM) were added into degradation system and incubated at 37 °C for 30 min. The MucB-L31W mutant (130 μM) significantly decreased the sensitivity to DDM even in a long-time incubation. In the degradation system, the concentrations of MucAperi, AlgW, and DDM are 125, 25 μM and 0.05% respectively.