Structural insight into the formation of lipoprotein-β-barrel complexes

Abstract

The β-barrel assembly machinery (BAM) inserts outer membrane β-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF–OMP complexes. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. RcsF is lodged deep within the lumen of the BamA barrel, binding regions proposed to undergo 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 following 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.

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Fig. 1: RcsF forms a complex with BamAB and BamABCDE.
Fig. 2: Structure of the BamA–RcsF complex.
Fig. 3: Conformational characteristics of the BamA–RcsF complex.
Fig. 4: BamA–RcsF is a proxy for the inward-open conformation of BamA.

Data availability

Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 6T1W. All other data generated or analyzed during this study are included in this published article and its Supplementary Information file. Source Data are provided with this paper.

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Acknowledgements

We thank A. Boujtat for technical help. We thank P. R. Chen (Peking University) for sharing DiZPK, H. Bernstein (NIH, Bethesda, USA) for providing strains and plasmids and M. Deghelt, G. Laloux, C. Goemans (EMBL, Heidelberg, Germany) and P. Leverrier for helpful suggestions and discussions, and for providing comments on the manuscript. We thank P. Legrand and staff at Soleil Synchrotron France and at Diamond Light Source UK for beamtime and their assistance during data collection. This work was supported, in part, by grants from the Fonds de la Recherche Scientifique (FNRS), from FRFS-WELBIO grants nos. WELBIO-CR-2015A-03 and WELBIO-CR-2019C-03, from the EOS Excellence in Research Program of the FWO and FRS-FNRS (no. G0G0818N), from the Fédération Wallonie-Bruxelles (no. ARC 17/22-087), from the European Commission via the International Training Network Train2Target (no. 721484), from the French region Ile-de-France (DIM Malinf) and from the BBSRC (nos. BB/P000037/1 and BB/M012573/1).

Author information

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Authors

Contributions

J.-F.C., R.R.A., S.E.R., H.R. and S.-H.C. wrote the manuscript. J.L., R.R.A., V.S.N., G.L., S.-H.C., H.R. and J.-F.C. designed the experiments. J.L., R.R.A., V.S.N., G.L. and S.-H.C. performed the experiments, constructed the strains and cloned the constructs. B.I.I. performed the dynamic importance sampling simulations. J.L., R.R.A., V.S.N., S.-H.C., H.R. and J.-F.C. analyzed and interpreted the data. A.N.C. and S.E.R. performed and analyzed the crosslinking–mass spectrometry experiments. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Seung-Hyun Cho or Han Remaut or Jean-François Collet.

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Extended data

Extended Data Fig. 1 RcsF can be co-purified with the BAM complex.

a,b,c, Gel filtration profiles of the affinity-purified BamAB-RcsF, BAM-RcsF and BAM complexes. The size exclusion chromatography was performed using a HiLoad 10/300 Superdex 200 pg. The input and peak fractions were collected and the samples were analyzed by blue native electrophoresis with Coomassie staining. The migration pattern of BamABCDE-RcsF (b) was modified compared to BamABCDE (c) upon size exclusion chromatography (band 8 increases), reflecting the higher instability of the BamABCDE-RcsF complex. n = 4 biologically independent experiments.

Extended Data Fig. 2 Crystal structure of the BamA-RcsF complex.

a, b, Final 2Fo-Fc electron map of the BamA-RcsF complex, shown with a map contour level of 0.08 e-/Å3 (root mean square deviation 1.02 Å). The asymmetric unit of the crystals holds two BamA-RcsF copies, one revealing interpretable electron density for the full BamA sequence (a), and a second revealing unambiguous density for POTRA domain 4 only (b). In the second copy (b), the electron density corresponding to POTRA domains 1, 2, 3, and 5 is too weak to allow unambiguous rigid body placement of the domains. All descriptions and images in the main text are based on the first copy (a). (c) Overlay of two BamA-RcsF complexes in the asymmetric unit. The first complex depicts BamA in gold and RcsF in blue, while these molecules are yellow and light blue, respectively, in the second complex. In both copies, RcsF makes an average displacement of 4 Å relative to the BamA β-barrel. (d) Crystal packing of the BamA-RcsF complex viewed along the a- (left) and c-axis (right). For the first copy of the BamA-RcsF complex in the asymmetric unit (orange-slate) the conformation of the POTRA domains is stabilized by the packing along the b-axis, whilst for the second copy (cyan-slate) only POTRA domain 4 is involved in crystal contacts. In the latter, POTRA 5, 3, 2 and 1 are not in contact with neighboring molecules and show weak electron density only due to the lack of conformational stabilization.

Extended Data Fig. 3 Structural dynamics of the BamA POTRA domains.

a,b, Superimposition of BamA-RcsF (gold and blue, respectively) with the POTRA domains in the inward-open BamABCDE complex (PDB: 5D0O; light blue) or the outward-open BamACDE complex (PDB: 5EKQ; green). Complexes are superimposed based on 400 equivalent Cα atoms in the BamAβ-barrel, and shown in side (a) or periplasmic (b) view. For 5d0o and 5ekq, the accessory Bam subunits and the BamA β-barrel are omitted for clarity. c, Periplasmic view of the inward-open BamABCDE complex, showing binding of the BAM accessory proteins BamB (magenta), BamC (red), BamD (blue), and BamE (yellow). Pulldown experiments showed that RcsF binds the BamABCDE complex (Fig. 1). In agreement with this observation, structural comparisons reveal that RcsF binding would not result in direct steric clashes with any BAM accessory protein. However, the positions of the POTRA domains in the BamA-RcsF and BamABCDE complexes are markedly different. In the BamA-RcsF complex, POTRA5 makes a 26° outward rotation to accommodate RcsF (see also Fig. 3), and a reorganization in the joint between POTRA domains 3 and 2 results in a more extended conformation of the POTRA ‘arm’ and the projection of POTRA domains 2 and 1 further from the BamA β-barrel, a conformation not previously reported in available BamA structures. In the BamABCDE complex, BamD contacts both POTRA5 and the joint of POTRA domains 1 and 2. In the BamA-RcsF complex, POTRA5 and POTRA domains 2 and 1 are too distant to be bridged by BamD; binding of BamD to BamA-RcsF therefore requires a conformational change in the POTRA arm or the dissociation of BamD at either of these two contact points.

Extended Data Fig. 4 Validation of the BamA-RcsF structure.

a, RcsF aminoacid sequence. The sequence coverage of the XL-MS experiment was about 60% as highlighted in violet b, Ribbon diagram of the BamA-RcsF structure. Highlighted residues show sites mutated to amber for incorporation of the photoreactive lysine analog DiZPK. Sites that crosslink to RcsF are green, sites that show no crosslinking are magenta. Mutation of extracellular loop 1 (eL1; red) leads to loss of RcsF binding (see panel g). BamA sidechains found to crosslink with RcsF by means of the homobifunctional amine-reactive crosslinker disuccinimidyl dibutyric urea (DSBU) are shown as sticks and colored cyan. Residue K61 from RcsF, which was found to crosslink to BamA using DSBU, is shown as a stick and colored orange. The other two RcsF residues (K42 and K134) that could be crosslinked to BamA are not visible in this structural model. c, In vivo photocrosslinking experiment in which cells expressing the BamA mutants containing DiZPK at the indicated positions were treated (+) or not (-) with ultraviolet light. Proteins samples were analyzed via SDS-PAGE and immunoblotted with anti-RcsF or anti-BamA antibodies, showing that the photo-crosslinked complexes contain BamA and RcsF. WT, wild type. d,e, Sensorgrams from biolayer interferometry (left) and corresponding equilibrium binding plots (right) of immobilized RcsF titrated with BamA (d) or immobilized BamA titrated with RcsF (e), n = 1 biologically independent experiment. f, The levels of major OMPs are slightly decreased in cells expressing BamA∆loop1. WT cells harboring the empty plasmid (pAM238) were used as control and EF-Tu expression levels were analyzed as loading control. n = 3 biologically independent experiments. g, Deletion of loop 1 in BamA prevents RcsF from being pulled down with BamA. WT cells harboring the empty plasmid (pAM238) were used as control. n = 3 biologically independent experiments. h, Overexpression of pBamAΔLoop1 in a bamA deletion strain activates the Rcs system compared to WT. A chromosomal rprA::lacZ fusion was used to monitor Rcs activity, and specific β-galactosidase activity was measured from cells at mid-log phase (OD600 = 0.5). Boxplot with whiskers from minimum to maximum. All values were normalized to the average activity obtained for WT cells harbouring the empty plasmid (pET3a) obtained from N = 8 biologically independent experiments. Mean is showed as +. WT, wild-type; Kan, kanamycin. Source data

Extended Data Fig. 5 RcsF binds the inward-open conformation of BamA.

a, Models for the BamAG393C/G584C 5 and BamAG433C/N805C 26 double cysteine mutants, which are locked in the outward-open or inward-open conformation, respectively, when oxidized. Mutated cysteines are shown as atom spheres. b, BamA barrel locking and RcsF binding. Overexpression of double cysteine mutants pBamAG393C/G584C-B and BamAG433C/N805C-B in a wild-type strain. RcsF can be co-purified with the BamA β-barrel locked in the inward-open conformation (BamAG433C/N805C) by a disulfide bond (ox) but not in the outward-open conformation (BamAG393C/G584C). BamA mutants become reduced (red) following treatment with tris(2-carboxyethyl) phosphine (TCEP) and migrate similarly. The oxidized form of BamAG393C/G584C migrates more slowly than wild-type BamA. As a result, two bands are visible for BamA in the input of BamAG393C/G584C, the lower migrating band corresponding to wild-type BamA expressed from the chromosome. n = 3 biologically independent experiments. c, Sensorgram from biolayer interferometry of immobilized RcsF titrated with BamAG393C/G584C, without (oxidized; - DTT) or with dithiothreitol (reduced; + DTT). When the β-barrel is locked in the outward-open conformation (- DTT), RcsF is unable to bind BamA. When reduced, BamAG393C/G584C regains binding, demonstrating that BamA reverts to the inward-open conformation in which it can bind RcsF. Source data

Extended Data Fig. 6 The movement of POTRA5 towards the periplasmic exit of the lumen of the BamA barrel could push RcsF upwards.

a,b, Lateral view of the initial and final conformations, respectively, of the BamA-RcsF complex during the dynamic importance sampling simulation (DIMS) of the BamA-RcsF complex. c,d, Bottom view (from the periplasm) of the above conformations. BamA and RcsF are colored in orange and blue, respectively. The initial conformation of the system (BamA and RcsF) corresponds to the structure determined in this work (PDB code 6T1W)5 with the POTRA1-4 domains removed. The final conformation of BamA is similar to the outward-open structure (PDB code 5D0Q). The explicit outer membrane and solvent are not shown for clarity. e, Expression from BamAhinge from a plasmid in ΔbamA cells leads to a severe growth defect when cells are grown at 37 °C in rich media, but not when they are grown in minimal media at 30 °C. Cells were grown in M9 minimal glucose medium at 30 °C until they reached OD600 = 1. Tenfold serial dilutions were made in M9 minimal glucose, plated onto M9 minimal glucose or LB agar, and incubated at 30 °C or 37 °C. Plates were supplemented with ampicillin (200 μg/ml). n = 3 biologically independent experiments.

Supplementary information

41589_2020_575_MOESM3_ESM.mov

DIMS simulation of the BamA–RcsF complex reproducing the proposed push-and-pull model. The simulation shows the transition of BamA from the inward-open to the outward-open conformation, with the POTRA5 domain moving towards the periplasmic exit of the lumen and pushing RcsF upwards. This movement is subsequently accompanied by the movement of strands 1–6 in BamA (Z1 domain) and the opening of the outward-facing extremity. The initial conformation of the system (BamA and RcsF) corresponds to the inward-open structure determined in this work (PDB: 6T1W) with the POTRA1–4 domains removed. The final conformation of BamA is similar to the outward-open structure (PDB: 5D0Q). The proteins are represented as cartoons (BamA and RcsF colored orange and blue, respectively), the explicit outer membrane is represented as sticks and the oxygen atoms of water molecules represented as red dots.

Supplementary Information

Supplementary Tables 1–5.

Reporting Summary

Supplementary Video 1

DIMS simulation of the BamA–RcsF complex reproducing the proposed push-and-pull model. The simulation shows the transition of BamA from the inward-open to the outward-open conformation, with the POTRA5 domain moving towards the periplasmic exit of the lumen and pushing RcsF upwards. This movement is subsequently accompanied by the movement of strands 1–6 in BamA (Z1 domain) and the opening of the outward-facing extremity. The initial conformation of the system (BamA and RcsF) corresponds to the inward-open structure determined in this work (PDB: 6T1W) with the POTRA1–4 domains removed. The final conformation of BamA is similar to the outward-open structure (PDB: 5D0Q). The proteins are represented as cartoons (BamA and RcsF colored orange and blue, respectively), the explicit outer membrane is represented as sticks and the oxygen atoms of water molecules represented as red dots.

Source data

Source Data Fig. 4b

Uncropped immunoblot images shown in Fig. 4b. a, Uncropped image of the anti-BamA, -BamB, -BamC, -BamD, -BamE and -RcsF immunoblot. The red box outlines the final cropped image. b, Uncropped image of the anti-Ef-Tu immunoblot. The red box outlines the final cropped image.

Source Data Extended Data Fig. 4f–g

Uncropped immunoblot images shown in Extended Data Fig. 4g. a, Uncropped image of the anti-BamA and -RcsF immunoblot. The red box outlines the final cropped image. b, Uncropped image of the anti-BamA, -BamB, -BamC, -BamD, -BamE and -RcsF immunoblot. The red box outlines the final cropped image. Uncropped immunoblot images shown in Extended Data Fig. 4g. a, Uncropped image of the anti-BamA and -RcsF immunoblot. The red box outlines the final cropped image. b, Uncropped image of the anti-BamA, -BamB, -BamC, -BamD, -BamE and -RcsF immunoblot. The red box outlines the final cropped image.

Source Data Extended Data Fig. 4h

Statistical Source Data for Extended Data Fig. 4h.

Source Data Extended Data Fig. 5b

Uncropped immunoblot images shown in Extended Data Fig. 5b. a, Uncropped image of the anti-BamA and -RcsF immunoblot. The red box outlines the final cropped image. b, Uncropped image of the anti-BamA and -RcsF immnoblot. The red box outlines the final cropped image, BamA bands after TCEP treatment. c,Uncropped image of the anti-BamB, -BamC, -BamD, -BamE and -RcsF immunoblot. The red box outlines the final cropped image.

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Rodríguez-Alonso, R., Létoquart, J., Nguyen, V.S. et al. Structural insight into the formation of lipoprotein-β-barrel complexes. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-020-0575-0

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