Folding of a bacterial integral outer membrane protein is initiated in the periplasm

Rakesh Sikdar,D Eric Anderson,Harris D Bernstein,Janine H Peterson

doi:10.1038/s41467-017-01246-4

Rakesh Sikdar, D Eric Anderson + Show 2 more

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https://doi.org/10.1038/s41467-017-01246-4

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The Bam complex promotes the insertion of β-barrel proteins into the bacterial outer membrane, but it is unclear whether it threads β-strands into the lipid bilayer in a stepwise fashion or catalyzes the insertion of pre-folded substrates. Here, to distinguish between these two possibilities, we analyze the biogenesis of UpaG, a trimeric autotransporter adhesin (TAA). TAAs consist of three identical subunits that together form a single β-barrel domain and an extracellular coiled-coil (“passenger”) domain. Using site-specific photocrosslinking to obtain spatial and temporal insights into UpaG assembly, we show that UpaG β-barrel segments fold into a trimeric structure in the periplasm that persists until the termination of passenger-domain translocation. In addition to obtaining evidence that at least some β-barrel proteins begin to fold before they interact with the Bam complex, we identify several discrete steps in the assembly of a poorly characterized class of virulence factors.

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The Bam complex promotes the insertion of β-barrel proteins into the bacterial outer membrane, but it is unclear whether it threads β-strands into the lipid bilayer in a stepwise fashion or catalyzes the insertion of pre-folded substrates
Early studies showed that periplasmic chaperones including Skp and SurA have important roles in the biogenesis of bacterial outer membrane (OM) proteins (OMPs)[3, 4], the discovery that a heterooligomer called the Bam complex is required for the membrane integration of β-barrels was a major breakthrough[5, 6]
By examining a wild-type version of UpaG and a mutant that fails to interact with the Bam complex, we find that the β-barrel assembles in the periplasm into an asymmetric trimer that persists even after the protein is integrated into the OM

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Introduction

The Bam complex promotes the insertion of β-barrel proteins into the bacterial outer membrane, but it is unclear whether it threads β-strands into the lipid bilayer in a stepwise fashion or catalyzes the insertion of pre-folded substrates. Most proteins that reside in the outer membrane (OM) of Gram-negative bacteria reach their destination post-translationally and contain a unique membranespanning segment known as a β-barrel This type of membranespanning segment is an amphipathic β-sheet that forms a closed cylindrical structure. The biogenesis of TAAs has been studied only superficially, and mutations that perturb assembly have been classified mainly by their effect on protein stability[25, 30,31,32] Based on their similarity to classical autotransporters it seems likely that the passenger domain is secreted in a C-to-N-terminal fashion through the formation of a hairpin structure[33,34,35]

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