A computational model for lipid-anchored polysaccharide export by the outer-membrane protein GfcD

Abstract

AbstractMany bacteria are protected by different types of polysaccharide capsules, structures formed of long repetitive glycan chains that are sometimes free and sometimes anchored to the outer membrane via lipid tails. One type, called group 4 capsule, results from expression of thegfcABCDE-etp-etkoperon inEscherichia coli. Of the proteins encoded in this operon, GfcE is thought to provide the export pore for free polysaccharide chains, but none of the proteins has been implicated in the export of chains carrying a lipid anchor. For this function, GfcD has been a focus of attention as the only outer-membrane β-barrel encoded in the operon. AlphaFold predicts two β-barrel domains in GfcD, a canonical N-terminal one of 12 strands and an unusual C-terminal one of 13 strands, which features a large lateral aperture between strands β1 and β13. This immediately suggests a lateral exit gate for hydrophobic molecules into the membrane, analogous to the one proposed for the lipopolysaccharide export pore LptD. Here, we report an unsteered molecular dynamics study of GfcD embedded in the bacterial outer membrane, with the common polysaccharide anchor, lipid A, inserted in the pore of the C-terminal barrel. Our results show that the lateral aperture does not collapse during simulations, that membrane lipids nevertheless do not penetrate the barrel, but that the lipid chains of the lipid A molecule readily exit into the membrane.Statement of SignificanceDespite the essential role polysaccharide capsules play in the resilience of bacteria to hostile environments, many aspects of their biogenesis are still poorly understood. One aspect concerns the export of capsular polysaccharides carrying a lipid anchor, for which even the proteins mediating the process are unknown. Here we propose that one of the largest families of β-barrel proteins in the bacterial outer membrane is a key agent of this process and show by biophysical simulation that it allows the exit of lipid anchors into the membrane through a lateral opening. More generally, our model illuminates the lateral exit mechanisms proposed for the export of hydrophobic macromolecules into the bacterial outer membrane.Graphical abstract