Structure and Function of the β-Barrel Assembly Machine and its Associated Chaperones

Abstract

Membrane insertion of beta-barrel Outer Membrane Proteins (OMPs) is essential for Gram-Negative bacteria and requires the coordinated action of both periplasmic chaperones and the outer membrane complex known as beta-Barrel Assembly Machinery (BAM). BamA, a beta-barrel OMP itself, is the central component of BAM, which also has four associated lipoproteins: BamB, C, D and E. BamA and BamD are essential for viability and appear to be present in all bacteria with an outer membrane. In the current model, periplasmic chaperones prevent OMP aggregation in the periplasm and deliver their cargo to the BAM complex for insertion into the outer membrane. We have determined the crystal structure of the periplasmic chaperone Skp. With NMR and crosslinking experiments we show that Skp binds the transmembrane domain of OmpA, a model beta-barrel OMP, in its cavity maintaining it in an unfolded state while protecting it from aggregation. Interestingly, the periplasmic, non-membrane domain of OmpA is allowed to fold to its native conformation explaining the specific importance of Skp on the OMP biogenesis pathway. We also present high-resolution structures of the lipoproteins BamB, C and D as well as the periplasmic domain of BamA, which contains five POTRA motifs thought to organize the BAM complex and associate with nascent OMPs en route to insertion in the outer membrane. In addition, we report the first structure of a complex between BamB and a POTRA3-5 fragment of BamA that begins to illuminate the BAM complex architecture. Using complementary Small Angle X-ray Scattering and NMR we show that the BamA POTRA domain undergoes conformational transitions, which functional experiments show are crucial for its role in OMP folding and insertion.