Bifurcated binding of the OmpF receptor underpins import of the bacteriocin colicin N into Escherichia coli.

Colicins are Escherichia coli-specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A colicins typically parasitize the proton-motive force-linked Tol system in the inner membrane via porins after first binding an outer membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that lipopolysaccharide (LPS) enhances this binding. Approximately 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A colicins.

Crystal structure of BamB bound to a periplasmic domain fragment of BamA, the central component of the ß-barrel assembly machine.

The ß-barrel assembly machinery (BAM) mediates folding and insertion of ß-barrel outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. BAM is a five-protein complex consisting of the ß-barrel OMP BamA and lipoproteins BamB, -C, -D, and -E. High resolution structures of all the individual BAM subunits and a BamD-BamC complex have been determined. However, the overall complex architecture remains elusive. BamA is the central component of BAM and consists of a membrane-embedded ß-barrel and a periplasmic domain with five polypeptide translocation-associated (POTRA) motifs thought to interact with the accessory lipoproteins. Here we report the crystal structure of a fusion between BamB and a POTRA3-5 fragment of BamA. Extended loops 13 and 17 protruding from one end of the BamB ß-propeller contact the face of the POTRA3 ß-sheet in BamA. The interface is stabilized by several hydrophobic contacts, a network of hydrogen bonds, and a cation-π interaction between BamA Tyr-255 and BamB Arg-195. Disruption of BamA-BamB binding by BamA Y255A and probing of the interface by disulfide bond cross-linking validate the physiological relevance of the observed interface. Furthermore, the structure is consistent with previously published mutagenesis studies. The periplasmic five-POTRA domain of BamA is flexible in solution due to hinge motions in the POTRA2-3 linker. Modeling BamB in complex with full-length BamA shows BamB binding at the POTRA2-3 hinge, suggesting a role in modulation of BamA flexibility and the conformational changes associated with OMP folding and insertion.

Crystal structure of BamB from Pseudomonas aeruginosa and functional evaluation of its conserved structural features.

The assembly of ß-barrel Outer Membrane Proteins (OMPs) in the outer membrane is essential for gram-negative bacteria. The process requires the ß-Barrel Assembly Machine (BAM), a multiprotein complex that, in E. coli, is composed of the OMP BamA and four lipoproteins BamB-E. Whereas BamA and BamD are essential, deletion of BamB, C or E produce membrane permeability defects. Here we present the high-resolution structure of BamB from Pseudomonas aeruginosa. This protein can complement the deletion of bamB in E. coli indicating that they are functionally equivalent. Conserved structural features include an eight-bladed ß-propeller fold stabilized by tryptophan docking motifs with a central pore about 8 Å in diameter at the narrowest point. This pore distinguishes BamB from related ß-propellers, such as quinoprotein dehydrogenases. However, a double mutation designed to block this pore was fully functional indicating that the opening is not essential. Two loops protruding from the bottom of the propeller are conserved and mediate binding to BamA. Conversely, an additional loop only present in E. coli BamB is not required for function. A cluster of highly conserved residues in a groove between blades 6 and 7 is crucial for proper BamB folding or biogenesis. It has been proposed that BamB may bind nascent OMPs by ß-augmentation to its propeller outer strands, or recognize the aromatic residue signature at the C-terminus of OMPs. However, Isothermal Titration Calorimetry experiments and structural analysis do not support these proposals. The structural and mutagenesis analysis suggests that the main function of BamB is to bind and modulate BamA, rather than directly interact with nascent OMPs.