Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

“…This study provided evidence that BamA forms a hybrid barrel with an incoming OMP through a stable interaction between β1 and the C-terminal β strand of the client (a segment that contains the conserved “β signal” motif GXXφXφ, where φ is an aromatic amino acid) ( 13 ) and a more dynamic interaction between β15/β16 and N-terminal β-strands of the client ( 28 ). Consistent with the biochemical data, a high-resolution structure of the same assembly intermediate bound to the Bam complex determined by cryo-EM confirmed both the interaction of BamA β1 with the β signal and the extreme dynamics of the interaction on the other side of the hybrid barrel ( 29 ). Although these studies strongly support an entirely new model in which the β signal first binds to BamA and then catalyzes the insertion of the remainder of the barrel by a “swing” mechanism, the data do not fully address the structure of the client protein at early stages before it is integrated into the OM.…”

Dynamic interplay between the periplasmic chaperone SurA and the BAM complex in outer membrane protein folding

“…OMP binding to SurA causes the OMP to populate expanded states 31 , 34 , 35 The OMP–SurA complex binds to BAM, to form an OMP–SurA–BAM ternary complex, resulting in conformational changes in both SurA (opening of the P1 and P2 domains) and BAM (favouring the lateral-closed state, which we propose to be the OMP acceptor state). The OMP binding cradle of SurA is oriented into the BAM periplasmic ring, allowing release of the unfolded OMP into a protective ‘chaperonin-like’ environment 5 , and presentation of the OMP to the BamA β-barrel for folding in a C- to N-terminus direction via β-strand elongation from β1 of the BamA barrel 5 , 52 , 53 , 59 , 61 , 114 . Here, a single β-hairpin of the substrate is depicted bound to β1 of the BamA barrel, however more extensive β-sheet structure in the substrate may begin to form in the periplasm (or at the water–membrane interface) prior to membrane integration 5 , 53 , 114 , 115 , in a manner analogous to a seeded amyloid aggregation reaction 5 , 116 , 117 .…”

“…Multiple lines of evidence suggest an allosteric connection between the periplasmic region of BAM and the BamA β-barrel domain including: (1) the correlation between lateral opening of the BamA β-barrel gate and a rotational conformational change in the BAM periplasmic ring in BAM structures 10 – 13 ; (2) a mutation in BamD (R197L) or deletion of BamE affects the surface exposure of BamA extracellular loop 6 (eL6) 94 , 95 ; (3) mutations in BamA eL6 suppress OMP assembly defects caused by the periplasmic BamA E373K mutation 96 ; (4) substitutions in the BamA β-barrel domain rescue the synthetic lethal phenotype of a ∆bamB∆bamE strain 97 ; and (5) mutations in the BamA β-barrel domain can overcome OMP assembly defects in BamA barrel-POTRA chimeras 98 . The combined evidence from structural studies suggests that the lateral-closed conformation of BamA (also known as ‘inward-open’), in which the BamA barrel is accessible from the periplasm, may be the acceptor state for receipt of unfolded OMPs 59 – 61 , 63 , 65 . Here, we have shown that protection from HDX occurs in the BamA barrel in the presence of SurA, suggesting that binding of the chaperone to the periplasmic region of BAM may favour the BamA lateral-closed state.…”

“…Models in which the substrate makes direct interactions with the BamA barrel have also been proposed 5 , 6 , 45 , 51 – 53 , and are supported by a variety of experimental evidence 54 – 56 , including in vivo crosslinking 53 , 57 , 58 and structural data 59 – 62 . For example, a cryoEM structure of a late-stage folding intermediate of BamA being folded by BAM 59 , as well as recent structures of EspP stalled while folding on BAM 61 , 62 , indicate that the terminal strand of an incoming OMP makes a β-augmentation interaction with the β1 strand of the BamA barrel as part of the assembly mechanism. Additional structures of BamA/BAM in which OMP-derived β-strands 60 , 63 or darobactin (a peptide antibiotic that targets BamA) 64 , 65 , are bound to β1 of the BamA barrel suggest that the lateral-closed state may be responsible for the receipt of unfolded OMPs for folding into the OM.…”

Detergent headgroups control TolC folding in vitro

“…To traverse the periplasmic space, OMPs are aided by periplasmic chaperones (6)(7)(8)(9). The folding of the majority of OMPs requires the BAM machinery which catalyzes insertion into the outer membrane (10)(11)(12)(13)(14).…”