Fig. 5. Hydrophilic and hydrophobic cavities participate in “trap-and-flip” of lipids.

a Vacuum electrostatic surface representation of inward-facing model of LtaA. Residues forming the hydrophobic and hydrophilic pockets are shown. b A model of a glycolipid molecule docked into the amphipathic cavity of LtaA. The lipid tail length corresponds to C16 chains. N-terminal and C-terminal domains are shown in light orange and light blue, respectively. c S. aureus cell growth on LB agar plates containing 0.1 mM IPTG, buffered at pH 6.4. The ΔltaA mutant is complemented with pLOW vector carrying a ltaA-WT gene or the annotated point mutations; Control vector indicates the pLOW vector carrying an unrelated gene (dCas9). d Mutagenesis analysis of the hydrophobic pocket. Relative flipping activity of LtaA-WT and variants. Error bars show + /− s.d. of technical replicates, n = 3. e Headgroup selectivity analysis. Relative flipping activity of LtaA in the presence of different concentrations of digalactosyldiacylglycerol (Gal₂DAG). Molar excess of Gal₂DAG over Glc₂-DAG-NBD is indicated. Error bars show + /− s.d. of technical replicates, n ≥ 3. Source data are provided as a Source Data file. f Proposed mechanism of LtaA catalyzed glycolipid transport. Schematic of conformational states throughout LtaA transport cycle.