Conformational dynamics and putative substrate extrusion pathways ofthe N-glycosylated outer membrane factor CmeC from campylobacter jejuni.

Abstract

The outer membrane factor CmeC of the efflux machinery CmeABC plays an important role in conferring antibiotic and bile resistance to Campylobacter jejuni. Curiously, the proteins in this machinery are N-glycosylated, with the glycans playing a key role in the effective function of the efflux system. Here we have employed atomistic equilibrium molecular dynamics simulations of CmeC in a representative model of the C. jejuni outer membrane to characterise the dynamics of the protein and its associated glycans. We show that the N-glycans are more conformationally labile than had previously been thought. The extracellular loops of CmeC visit the open and closed states freely suggesting the absence of a gating mechanism on this side, while the narrow periplasmic entrance remains tightly closed, regulated via coordination to solvated cations. We identify several cation binding sites on the interior surface of the protein. Additionally, we have used steered molecular dynamics simulations to elucidate translocation pathways for a bile acid, chenodeoxycholic acid, and a macrolide antibiotic, erythromycin. These, and additional equilibrium simulations suggest that the anionic bile acid utilizes multivalent cations to climb a ladder of acidic residues that line the interior surface of the protein. In combination, these results further our understanding of the dynamics of outer membrane factors and allow insight into the translocation pathways of antibiotics and other known substrates in this region of the efflux machinery.