A Comparison of in vivo and in vitro Bama Barrel Seam Dynamics

Abstract

The outer membrane (OM) of Gram-negative bacteria is essential for cell growth and survival. Outer membrane proteins (OMPs) have a β-barrel structure and are responsible for carrying out a number of cellular processes such as nutrient uptake and interaction with the host. OMPs are synthesized in the cytoplasm and are transported to the OM where they are folded and inserted by the β-barrel assembly machine (BAM) complex. Although the role of the BAM complex is known, its mechanism of action remains a mystery. BamA is the central component of the BAM complex. It contains an N-terminal polypeptide-transport-associated (POTRA) domain and a C-terminal barrel domain. Crystal structures of BamA have indicated that the barrel seam, the interface between the first and last strands, is destabilized. This suggests that the barrel seam is involved in the mechanism of action. Indeed, it has been suggested that the seam functions as a lateral gate that opens and serves as a scaffold for OMP folding and that insertion happens via a ‘budding’ mechanism. Furthermore, disulfide locking of the barrel seam is lethal in Escherichia coli. However, previous work has shown that disulfide locking of the barrel does not prevent BAM from catalyzing in vitro folding of model OMPs into liposomes. Using disulfide trapping experiments, we show that the BamA barrel seam is highly dynamic in in vitro liposome membranes, undergoing 14 Å register shifts up and down the membrane. Furthermore, these seam dynamics are also observed in vivo in native E. coli outer membranes. Our findings define the remarkable range of motions populated by the BamA β-barrel in its native context.