Structural Characterization of Outer Membrane Protein Folding Intermediates

Abstract

Transmembrane proteins with a β-barrel topology are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. These proteins are folded by a conserved protein complex termed the β-barrel assembly machine (BAM). Structural evidence has demonstrated that the central component of the complex, BamA – itself a β-barrel, interacts with substrates via β-augmentation. However, the mechanism by which BAM allows a substrate to fold is unclear, and what features of the machine allow it to effectively fold many different substrate barrels of varying size and shape is unknown. First, we develop a disulfide crosslinking assay that we use to identify folding intermediates of substrate barrels as they are assembled on BAM. We use this assay to show that two β-barrel substrates, BamA and LptD, pass through multiple shared intermediates during their folding on the BAM complex. Next, we structurally characterize three of these sequential folding intermediates of a BamA barrel substrate folding on BAM using cryo-electron microscopy. This “movie” of snapshots shows that β-strands are added to the nascent substrate barrel from a disordered state within the machine BamA lumen. The snapshots also show how the machine BamA barrel variably distorts according to the stage of folding of a single barrel, and suggest that a substrate’s efficient release from the machine requires a properly ordered global substrate architecture. iv Finally, we structurally characterize two large substrate barrels, LptD and FimD, in the process of folding on the BAM complex. These structures suggest that BamA barrel distortion and β-templating of substrate strands from the BamA lumen are general folding features. They also present twists on this general folding mechanism, and allow us to propose models for how BAM folds large barrels which contain soluble plug domains. Together, these results provide a detailed structural picture of outer membrane protein folding, show how substrates grow via sequential addition of β-strands, and suggest that BAM’s ability to fold many different barrels is rooted in its ability to variably distort, allowing a substrate to find its thermodynamic minimum structure no matter the substrate barrel’s ultimate shape.Chemistry and Chemical Biolog