Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous β-barrel with impaired β1-β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.

The carbon sensor SbtB perceives diurnal oscillation of c-di-AMP to control glycogen synthesis and nighttime survival.

In Escherichia coli, a multicomponent BAM (␤-barrel assembly machinery) complex is responsible for recognition and assembly of outer membrane ␤-barrel proteins. The functionality of BAM in protein biogenesis is mainly orchestrated through the presence of two essential components, BamA and BamD. Here, we present crystal structures of four lipoproteins (BamB-E). Monomeric BamB and BamD proteins display scaffold architectures typically implied in transient protein interactions. BamB is a ␤-propeller protein comprising eight WD40 repeats. BamD shows an elongated fold on the basis of five tetratricopeptide repeats, three of which form the scaffold for protein recognition. The rod-shaped BamC protein has evolved through the gene duplication of two conserved domains known to mediate protein interactions in structurally related complexes. By contrast, the dimeric BamE is formed through a domain swap and indicates fold similarity to the ␤-lactamase inhibitor protein family, possibly integrating cell wall stability in BAM function. Structural and biochemical data show evidence for the specific recognition of amphipathic sequences through the tetratricopeptide repeat architecture of BamD. Collectively, our data advance the understanding of the BAM complex and highlight the functional importance of BamD in amphipathic outer membrane ␤-barrel protein motif recognition and protein delivery.Gram-negative bacteria are surrounded by two membranes, an inner membrane and the protective outer membrane (OM) 2 layer. The outer membrane architecture is highly asymmetric and composed by integral outer membrane ␤-barrel proteins, lipoproteins, lipopolysaccharides, and phospholipids (1). These components are entirely synthesized in the cytoplasm, translocated over the inner membrane, and finally delivered through the periplasm to the outer membrane by specific shuttle factors that transport their cargo to membrane receptor complexes (1, 2). In Escherichia coli, lipoproteins are targeted to the OM through the LolABCDE complex system via a series of membrane and periplasmic transfer steps (3, 4). Integral OMPs are translocated by the secretion (Sec) machinery and stabilized against premature precipitation or mislocalization in the periplasm by the three major chaperones PpiD, Skp, and SurA (4 -7). These chaperones presumably act sequentially with PpiD at an early stage, early after the OMP release into the periplasm (5). This initial rescue event is followed by Skp and SurA chaperoning at a later stage (8). Various experimental studies describing OMP folding through SurA show the particular importance of this chaperone. Phage display combined with peptide binding studies indicated SurA interactions with amphipathic OMP peptides (9, 10). Furthermore, the importance of SurA in vivo is underlined by the observation that E. coli cells being deleted in surA exhibit reduced levels of folded OmpA, OmpC, OmpF, and LamB porins (11).After the unfolded outer membrane protein is delivered through the periplasmic space, the final steps are the tetherin...

Most core residues of coiled coils are hydrophobic. Occasional polar residues are thought to lower stability, but impart structural specificity. The coiled coils of trimeric autotransporter adhesins (TAAs) are conspicuous for their large number of polar residues in position d of the core, which often leads to their prediction as natively unstructured regions. The most frequent residue, asparagine (N@d), can occur in runs of up to 19 consecutive heptads, frequently in the motif [I/V]xxNTxx. In the Salmonella TAA, SadA, the core asparagines form rings of interacting residues with the following threonines, grouped around a central anion. This conformation is observed generally in N@d layers from trimeric coiled coils of known structure. Attempts to impose a different register on the motif show that the asparagines orient themselves specifically into the core, even against conflicting information from flanking domains. When engineered into the GCN4 leucine zipper, N@d layers progressively destabilized the structure, but zippers with 3 N@d layers still folded at high concentration. We propose that N@d layers maintain the coiled coils of TAAs in a soluble, export-competent state during autotransport through the outer membrane. More generally, we think that polar motifs that are both periodic and conserved may often reflect special folding requirements, rather than an unstructured state of the mature proteins.ion coordination ͉ protein export ͉ trimeric autotransporter adhesin ͉ polar core residues

Diffuse retinal irradiation by visible light produces in the rat the death of visual cells and pigment epithelium. Typically, cage illumination of 1500 lux from fluorescent light through a green filter leads to severe damage when continued for 40 hours. Vitamin A deficiency protects against this damage but experiments show that retinol released by light from rhodopsin is probably not the toxic agent. Protection against light damage depends on a long-range state of cell adaptation to light itself. The normal diurnal cycle of light and dark seems to be the essential factor in controlling visual cell viability and susceptibility.