Inward-facing glycine residues create sharp turns in ß-barrel membrane proteins.

The transmembrane region of outer-membrane proteins (OMPs) of Gram-negative bacteria are almost exclusively ß-barrels composed of between 8 and 26 ß-strands. To explore the relationship between ß-barrel size and shape, we modeled and simulated engineered variants of the Escherichia coli protein OmpX with 8, 10, 12, 14, and 16 ß-strands. We found that while smaller barrels maintained a roughly circular shape, the 16-stranded variant developed a flattened cross section. This flat cross section impeded its ability to conduct ions, in agreement with previous experimental observations. Flattening was determined to arise from the presence of inward-facing glycines at sharp turns in the ß-barrel. An analysis of all simulations revealed that glycines, on average, make significantly smaller angles with residues on neighboring strands than all other amino acids, including alanine, and create sharp turns in ß-barrel cross sections. This observation was generalized to 119 unique structurally resolved OMPs. We also found that the fraction of glycines in ß-barrels decreases as the strand number increases, suggesting an evolutionary role for the addition or removal of glycine in OMP sequences.

BamA is required for autotransporter secretion.

BACKGROUND: In Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the "passenger" domain) and a ß-barrel that aids its export. While it is known that the folding and insertion of the ß-barrel domain utilize the ß-barrel assembly machinery (BAM) complex, how the passenger domain is secreted and folded across the membrane remains to be determined. The hairpin model states that passenger domain secretion occurs independently through the fully-formed and membrane-inserted ß-barrel domain via a hairpin folding intermediate. In contrast, the BamA-assisted model states that the passenger domain is secreted through a hybrid of BamA, the essential subunit of the BAM complex, and the ß-barrel domain of the autotransporter. METHODS: To ascertain the models' plausibility, we have used molecular dynamics to simulate passenger domain secretion for two autotransporters, EspP and YadA. RESULTS: We observed that each protein's ß-barrel is unable to accommodate the secreting passenger domain in a hairpin configuration without major structural distortions. Additionally, the force required for secretion through EspP's ß-barrel is more than that through the BamA ß-barrel. CONCLUSIONS: Secretion of autotransporters most likely occurs through an incompletely formed ß-barrel domain of the autotransporter in conjunction with BamA. GENERAL SIGNIFICANCE: Secretion of virulence factors is a process used by practically all pathogenic Gram-negative bacteria. Understanding this process is a necessary step towards limiting their infectious capacity.