Structural Basis For The Interaction Of BamB And POTRA Domains Of BamA

Secreted proteins (such as enzymes in signal transduction,virulence factors in pathogenic bacteria or antibody in immune-enhancing effect, etc.) are the main tools that bacteria use to interact with their environment. Eight major protein secretion systems have been discovered so far and named from type I secretion system to type VIII secretion system.In Gram-negative bacteria, various kinds of outer membrane proteins (OMPs) utilize Type V secretion system to secrete into outer membrane to maintain bacterial physiological functions, including bacterial communication, nutrient, uptake, immunity, pathogenicity, etc. Type V secretion system consists of two-step secretion pathways. First, these unfolded OMPs are dependent on SecYEG complex to transport across the inner membrane removing the signal peptide. Subsequently the OMPs are folded and integrated into outer membrane by the assistance of BAM (β-barrel assembly machinery) complex.In Escherichia coli, BAM complex is essential for outer membrane protein assembly. Recent studies have shown that the BAM complex includes the integral β-barrel protein BamA and the lipoproteins BamB, BamC, BamD and BamE.In the complex, BamA is the central component protein, which is responsible for folding and inserting OMPs into the outer membrane with the assistance of other components. BamB and BamD are crucial for BamA folding and biogenesis in vivo. However, E.coli lacking of BamB, BamC, or BamE exhibit defective in the outer membrane proteins assemble.So far, in the BAM complex, several structures of apo-BamB, BamC, BamD, BamE and BamCD have been determined. Based on biochemical analysis, BamA POTRA domains provide a scaffold to mediate BamBCDE interactions. BamA directly interact with BamB, forming a subcomplex. BamC and BamE are mediated by BamD to indirectly binding BamA. The two purified subcomplexes BamAB and BamCDE could be reconstituted holo BAM complex in vitro. From mutagenesis analysis, POTRA5 is required for BamCDE interactions and POTRA3-5 is important for E.coli viability. Moreover, POTRA3-5 is necessary for BamB binding. But the definite interactions of BAM complex are also elusive.Here, we present the crystal structure of BamB in complex with POTRA (polypeptide-transport-associated) domains of BamA at 2.1 A resolution. POTRA domains are associated with BamB via hydrogen bonding and hydrophobic interactions, which are contributed by β1,β2, β3,β2-loop-β3 in POTRA3 and DA, BC loops in BamB. Furthermore, POTRA3 domain is necessary and sufficient for BamB binding based on our crystal and in vitro binding studies. Structural and biochemical analysis reveal that the conserved residues Arg77, Glu127, Glu150, Ser167 and Arg195 of BamB play an essential role in the interaction with POTRA3 domain.