Fig. 8. Integration of a proposed lipid-modulated molecular assembly mechanism of nBamA with its folding in vivo. (A) In Gram-negative bacteria, periplasmic holdases such as Skp/SurA (gray) bind to and transport newly synthesized nBamA (maroon) across the periplasm to the BAM complex in the OM. BamA folding is nucleated locally in its Skp/SurA-bound state, prior to its handover to the pre-assembled BAM complex (PDB ID: 7BNQ; BamA^M in cerulean). BamD can also bind to an internal signal in nBamA and promote β-strand formation.⁷⁸ (B) Recruitment of BamA to the POTRA/BamB/C/D/E periplasmic ring simultaneously triggers local disruption of the OM and sliding of BamA^M β1 from β16. The latter step converts the “inward open” state (BamA^M β1 and β16 still H-bonded) of BamA^M (A) to the “outward open” state (β1 of BamA^M available to H-bond with the substrate) (B). Together, these changes energetically favor nBamA insertion. (C) Lateral gate opening of BamA^M (“gate wide open” state; shown here without the lipoproteins), where the N-terminal β-strands swing outwards, providing access to nBamA for its C → N directed membrane insertion. Our findings specifically show that this is thermodynamically facilitated by intrinsic destabilization of the N-terminal strands of BamA^M, particularly in PE-rich membranes where the BAM complex is highly active. (D/E) Proposed stepwise events in BamA β-strand formation and membrane integration, regulated by its immediate lipidic environment (based on our Φ-value analysis and proposed unassisted folding mechanisms we deduced; see Fig. 6 and 7). BamA can undergo β-augmentation in PE-rich membranes (D), while prefolded BamA swings into the OM in a PG-rich environment (E). (D) The “gate wide open” BamA^M in PE is less stable and highly dynamic at the N-terminal strands. First, nBamA β15–β16 threaded into the BamA^M lumen, forms stable H-bonding with β1 of BamA^M. BamA then undergoes β-strand elongation resembling the β-augmentation model.^74,79 The relatively slower folding of BamA in the presence of PE (D), coupled with SurA/Skp assistance, POTRA-mediated hand-off, and BAM complex assistance, can ensure that (i) unfolded protein in the periplasm does not insert into the inner membrane, which is also PE-rich, (ii) off-pathway irreversible structures in the membrane are not adopted after SurA/Skp hand-off, and (iii) the assembly is completed correctly after binding of lipoproteins. (E) In PG-rich local environments, a conformationally restricted BamA^M can promote nBamA structuring primarily in the periplasmic ring, membrane interface, and in the POTRA-lipoprotein-bound states. Interaction of nBamA with BamA^M-β1 occurs as one of the last events during folding after near-complete β-strand formation. β-Barrel assembly at the C-terminal and mid-barrel zones trigger membrane insertion of BamA following a swing mechanism.³⁴ In PG, BamA^M could primarily assist in β7–β8 assembly, wherein membrane translocation of L4 presents an energy barrier and occurs late in the folding process. We propose that the non-native interactions we observed at the N-terminus of BamA correspond to regions where nBamA is in contact with BamA^M during assembly, as seen in the lumen-catalyzed model.⁷⁶ (F) Membrane insertion of all the β-strands of BamA and hybrid nBamA–BamA^M formation (PDB ID: 6V05) eventually triggers β-barrel closure through β1–β16 association (intra-barrel H-bonding) of the respective polypeptides, ultimately releasing the newly formed BamA from BamA^M and into the membrane (G). All TS structures were rendered in PyMOL and assembled using CorelDraw.