The primer pairs were SDM-HisBamA F with SDM-HisBamA R and bamE delHis F with bamE delHis R

The primer pairs were SDM-HisBamA F with SDM-HisBamA R and bamE delHis F with bamE delHis R. with the lateral gate to the membrane closed. RcsF is lodged deep inside the lumen of the BamA barrel, binding regions proposed to undergo an outward and lateral opening during OMP insertion. On the basis of our structural and biochemical data, we propose a push-and-pull model for RcsF export upon conformational cycling of BamA and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity. Introduction The vast majority of proteins inserted in the outer membrane of Gram-negative bacteria adopt a -barrel conformation. Their assembly depends on the activity of the conserved -barrel assembly machinery (BAM), whose core component is the OMP85-family protein BamA1,2. BamA is an outer membrane 16-stranded -barrel with a large periplasmic extension consisting of five POlypeptide TRansport-Associated (POTRA) domains at its N-terminus1. Structures of BAM have shown that BamA can adopt two conformations: an outward-open conformation3,4, in which the -barrel domain opens between strands 1 and 16 to form a lateral gate to the membrane, and an inward-open conformation5,6, in which the lateral gate is sealed while a periplasmic entry pore to the barrel lumen is open. In the bacterium crosslinking experiments in which a complex between RcsF and BamA, considered to be an intermediate in the formation of RcsF-OmpA/C/F complexes, was trapped10,11. Further, in cells lacking BamB and BamE, RcsF accumulates on BamA and causes a lethal block to BAM-mediated OMP assembly, suggesting that OMPs and surface-exposed RcsF exploit at least partially overlapping assembly routes12,13. HMN-176 RcsF functions as an envelope stress sensor capable of mounting a protective response when damage occurs in the peptidoglycan or in the outer membrane14,15. Interestingly, we previously determined that sending RcsF to the surface is part of a cellular strategy that enables RcsF to detect damage in the cell envelope. Under stress conditions, newly synthesized RcsF molecules fail to interact with HMN-176 BamA10: they are not exported to the surface and remain exposed to the periplasm, which allows them to trigger the Rcs signaling cascade by reaching the downstream Rcs partner in the inner membrane16. Thus, surface exposure is intimately linked to the function of RcsF. However, the molecular details of the BamA-RcsF interaction, how BAM orchestrates the export of RcsF with OMP assembly, and what prevents RcsF from interacting with BamA under stress conditions remain unknown. Here we sought to address these questions by obtaining structural information about the interaction between BamA and RcsF. Results RcsF can be purified with the BAM complex In a series of exploratory experiments, we co-overexpressed RcsF with the BamAB sub-complex, or with the BamABCDE holocomplex; both BamAB-RcsF and BamABCDE-RcsF could be detergent-extracted from the membrane and purified via affinity chromatography using a His-tag on the N-terminus of BamA (Fig. 1a). Using native gel electrophoresis, we confirmed that RcsF binds BamABCDE, and not only BamAB (Fig. 1a, b, c). However, HMN-176 whereas BamAB-RcsF was stable and could be purified to homogeneity by size-exclusion chromatography, BamABCDE-RcsF was unstable (Extended Data Fig. 1a, b). Interesting to note, destabilization of BamABCDE was only observed when RcsF was present (Extended Data Fig. 1c). Open in a separate window Figure 1 RcsF forms a complex with BamAB and BamABCDE. (a, b) SDS-PAGE (a) and blue native (b) analysis of purified BAM, BAM-RcsF and BamAB-RcsF complexes obtained via BamA-affinity chromatography. The bands analyzed in (c) are labelled 1 to 8. (c) SDS-PAGE analysis of the complexes shown in panel b (bands 1 to 8). The BAM HMN-176 complex expressed from pRRA1 is a mixture of BamABCDE FAM162A and BamABDE. n= 4 biologically independent experiments. BamA is in the inward-open conformation in the structure The BamAB-RcsF complex was crystallized and its structure solved to 3.8 ? resolution by molecular replacement using the structures of BamA and RcsF (PDB: 5D0O and 2Y1B, respectively; Supplementary Table 1). While this structure contained BamA and RcsF (Fig. 2), BamB dissociated from the BamA-RcsF complex during crystallization and was absent. The.