LAUSR

Gram-negative bacteria possess two cell membranes. The outer membrane is host to almost exclusively β-barrel transmembrane proteins. The β-barrel assembly machinery (BAM) is a five-protein complex responsible for the insertion and assembly of outer membrane β-barrel proteins. The first crystal structures of the transmembrane β-barrel domain of BamA were released in 2013. In these initial crystal structures, the BamA β-barrel possessed a weak interface between its first (1) and last (16) β strands, which led to strand separation within 1 μs in our equilibrium simulations. This strand separation has been proposed to act as a lateral gate for substrate passage into the membrane. In addition, a hydrophobic mismatch near the barrel seam was shown to destabilize the membrane, potentially acting to aid integration of the substrate. Furthermore, full BAM complex crystal structures were recently released, showing accessory proteins rallying around this putative insertion region using the periplasmic domains of BamA as a scaffold. In order to address remaining questions surrounding the role that BamA plays in the insertion and assembly process, we have carried out additional equilibrium simulations of BamA in several membrane bilayers. We also performed a calculation of energetic landscapes associated with lateral gate formation under various conditions. These calculations reveal a lower energetic barrier to strand separation for BamA as compared to FhaC, a member of the same (Omp85) family. They also reveal a significant role played by the C-terminal kink feature in modulating the barrier to strand separation. Finally, equilibrium simulations of NgBamA demonstrate lateral gate opening at 340K in a native lipopolysaccharide bilayer for the first time.