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Skp is a multivalent chaperone of outer-membrane proteins

Abstract

The trimeric chaperone Skp sequesters outer-membrane proteins (OMPs) within a hydrophobic cage, thereby preventing their aggregation during transport across the periplasm in Gram-negative bacteria. Here, we studied the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding revealed that higher Skp/OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry–mass spectrometry (IMS–MS) data, computer modeling and molecular dynamics simulations provided evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs that cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp in protecting a single substrate from aggregation.

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Figure 1: Three-dimensional structures of Skp and the OMPs used in this study.
Figure 2: Different Skp/OMP ratios are required to sequester OMPs and inhibit folding.
Figure 3: Skp–OMP complexes have different stoichiometries.
Figure 4: Collision cross-section distributions of Skp and Skp–OMP complexes.
Figure 5: Possible architectures of Skp–OMP complexes.
Figure 6: In vacuo MD simulations of 1:1 and 2:1 Skp–OMP complexes.
Figure 7: MD simulations of 1:1 and 2:1 Skp–OMP complexes in explicit solvent.

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Acknowledgements

The plasmids containing the mature sequences of tOmpA, PagP, OmpA and BamA were kindly provided by K. Fleming (John Hopkins University)25. Plasmid pET21b (Novagen) containing the Skp-encoding gene was a gift from J. Bardwell (University of Michigan). We also thank S. Hiller (University of Basel) for kindly providing the His-tagged Skp construct15 and S. Buchanan (NIH) for the gift of plasmid BamAB-pETDUET-1. We thank T. Watkinson (University of Leeds) for expression and purification of OmpT and M. Jackson (University of Leeds) for help with western blotting, and we are also grateful for the assistance and advice of L.M. McMorran (University of Leeds) in the early stages of this work. This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/J014443/1 (B.S.), BB/K000659/1 (A.N.C.) and BB/J011819/1 (P.W.A.D.). Funding from the European Research Council under the European Union's Seventh Framework Programme grant FP7.2007-2013/grant agreement number 322408 (A.E.A., D.J.B. and S.E.R.) is also acknowledged. The Waters Synapt G1 and G2-Si mass spectrometers were purchased with funding from the BBSRC (BB/E012558/1 and BB/M012573/1, respectively). This project made use of time on the ARC2 supercomputer facility at the University of Leeds and time on ARCHER granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://www.hecbiosim.ac.uk/), supported by the EPSRC (grant no. EP/L000253/1).

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Authors and Affiliations

Authors

Contributions

B.S. and A.N.C. contributed equally to this work. B.S. designed and performed the kinetic experiments, computer modeling and MD simulations. A.N.C. designed and performed the MS and cross-linking experiments. P.W.A.D. designed and performed in vacuo apo-Skp simulations. S.A.H. assisted and provided supervision in the MD simulations. A.E.A., D.J.B. and S.E.R. conceived, designed and supervised the research. All authors contributed to the discussion and were involved in editing the final manuscript.

Corresponding authors

Correspondence to Alison E Ashcroft, David J Brockwell or Sheena E Radford.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 OMP folding transients in the absence of lipids.

Example kinetic traces for (a) tOmpA, (b) PagP, (c) OmpF, (d) tBamA and (e) OmpT, in the absence of lipids, monitored by fluorescence emission spectroscopy. Assays were performed with OMP concentrations of 0.4 µM, in 0.24 M urea, 50 mM glycine-NaOH, pH 9.5, at 25 ºC. At least three replicates are shown for each protein. (f) Crystal structure of OmpT, PDB: 1I78 (Vandeputte-Rutten, L. et al. EMBO J 20 5033-9, 2001). Tryptophan residues are shown in stick representation and highlighted in red. The data for OmpT are well described by a single exponential indicated by black dashed lines.

Supplementary Figure 2 16-stranded OMPs, compared with 8-stranded OMPs, require preincubation with a greater molar excess of Skp to inhibit folding into synthetic liposomes.

Example kinetic folding data for (a) tOmpA, left panel and tOmpA in the presence of a 1:1, centre panel, or 2:1, right panel, molar ratio of Skp:tOmpA; (b) PagP, left panel and PagP in the presence of a 1:1, centre panel, or 2:1, right panel, molar ratio of Skp:tOmpA; (c) OmpF, left panel and OmpF in the presence of a 2:1, centre panel, or 4:1, right panel, molar excess of Skp; (d) tBamA, left panel and tBamA in the presence of a 2:1, centre panel, or 4:1, right panel, molar excess of Skp. Pre-incubated Skp-OMP complexes were added to diC11:0PC liposomes and OMP folding was monitored by fluorescence spectroscopy. Final OMP concentrations were 0.4 µM, with a molar lipid:protein ratio of 3200ː1, in 0.24 M urea, 50 mM glycine-NaOH, pH 9.5. A minimum of three transients are shown in each panel. Single or double exponential fits to the data are indicated by black dashed lines (see Supplementary Table 2).

Supplementary Figure 3 Complexes of Skp with OMPs have variable stoichiometries, as revealed by ESI–IMS–MS.

IMS driftscope plots of (a) Skp and Skp:OMP complexes with (b) tOmpA, (c) PagP, (d) OmpT, (e) OmpF or (f) tBamA. Peaks are labelled with their charge state. Charge states corresponding to Skp, 1:1 Skp:OMP and 2:1 Skp:OMP are labelled in red, green and blue, respectively. (g) CCS distributions (peak heights normalized to MS peak intensity) of 2:1 Skp:OMP complexes with OmpT (left), OmpF (middle), and tBamA (right) obtained from ESI-IMS-MS analyses. The charge state for each CCS distribution is indicated.

Supplementary Figure 4 ESI–MS shows that two copies of Skp bind to full-length BamA.

(a) Non-covalent ESI mass spectrum of full-length BamA binding to Skp. The spectrum is annotated with yellow circles (BamA), red circles (Skp), green squares (1:1 Skp:BamA) and blue triangles (2:1 Skp:BamA). The most abundant charge state in each distribution is labelled. (b) IMS driftscope plot corresponding to the mass spectrum in (a). Charge states corresponding to Skp, BamA, 1:1 Skp:BamA, and 2:1 Skp:BamA are labelled in red, yellow, green and blue, respectively. The ions at m/z ~2000 arise from Skp subunits.

Supplementary Figure 5 Chemical cross-linking and SDS–PAGE, western blotting and MS analysis of Skp–OMP complexes.

(a) SDS-PAGE analysis of a mixture of Skp and OmpA incubated without (-) or with (+) cross-linker. Upon addition of cross-linker a new band appears at approx. 85 kDa, consistent with a cross-linked Skp:OmpA complex (Skp:OmpA: 89.3 kDa, Skp: 54.0 kDa, OmpA: 35.3 kDa). An additional band (triangle) may correspond to (Skp:OmpA)2 (178.6 kDa), likely due to dimerization via the periplasmic domain as previously observed (Marcoux, J. et al. Structure 22 781-790, 2014). (b) Western blotting analysis with an anti-His antibody to confirm the location of His-tagged Skp-containing bands in the cross-linked Skp:OmpA sample. Higher molecular weight bands in the cross-linked sample (> 250 kDa) are therefore likely due to intermolecular OmpA cross-links. (c-d) Peptides identified from excised and digested bands from SDS-PAGE gels of cross-linked Skp:OmpA complexes consistent with (c) a 1:1 Skp:OmpA complex (the molecular weight band at ~90 kDa in a,b), (d) a larger Skp:OmpA complex (the molecular weight band labelled with a triangle in a,b). (e) SDS-PAGE analysis of a mixture of Skp and BamA incubated without (-) or with (+) cross-linker. Note that upon addition of cross-linker, no band appears at the expected mobility for a 1:1 Skp:BamA complex (*, 142.5 kDa). Instead, a single high molecular weight band is observed which corresponds to a higher order Skp:OMP assembly, consistent with the 2:1 Skp:BamA complex observed by ESI-MS (196.5 kDa). (f) Western blotting analysis with an anti-His antibody showing the location of His-tagged Skp-containing bands in the cross-linked Skp:BamA sample. Higher molecular weight bands in the cross-linked sample in (e) (> 250 kDa) are therefore likely due to intermolecular BamA cross-links. (g) Peptides identified from the excised and digested band of a cross-linked Skp:BamA complex. Regions of Skp or the corresponding OMP identified by database searching in (c, d, g) are denoted in bold with grey shading.

Supplementary Figure 6 Molecular dynamics simulations of Skp in vacuo.

(a) Theoretical CCS, and (b) backbone RMSD (red line) and radius of gyration (Rg) (light blue line) calculated for the initial 10 ns of a 100 ns molecular dynamics simulation in the gas-phase. (c) The starting model Skp structure used for the MD simulation (PDB: 1U2M (Walton, T.A. & Sousa, M.C. Mol Cell 15 367-74, 2004), with missing residues in chains B and C modelled from chain A) (Supplementary Data Set 1). Skp subunits are colored green, blue and yellow. (d,e) Structures of Skp after a simulation time of (d) 0.2 ns and (e) 10 ns (Supplementary Data Set 2). The CCS values of the collapsed Skp structures after a simulation time of 10 ns and 100 ns (38.0 ± 1.8 nm2 and 37.3 ± 1.9 nm2, respectively) (see also Supplementary Table 5) agree favorably with the modal CCS of Skp at the lowest observed charge state (37.9 ± 0.6 nm2) in native-MS experiments (Fig. 4a).

Supplementary Figure 7 Molecular dynamics simulations of Skp in solvent.

(a) Starting structure used for explicit solvent MD simulation of Skp (PDB: 1U2M3, with missing residues in chains B and C modelled from chain A), shown from the side (left) and bottom (right) (Supplementary Data Set 1). (b) Example structure of Skp in an ‘open’ conformation (t = 7.5 ns), shown from the side (left) and bottom (right) (Supplementary Data Set 3). (c) Radius of gyration of Skp over the course a MD simulation in explicit solvent. Structural collapse of initially extended chains of (d) tOmpA and (e) tBamA simulated with an implicit solvent model (see Online Methods).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–5 (PDF 1455 kb)

Supplementary Data Set 1

Starting model of Skp used in MD simulations (TXT 520 kb)

Supplementary Data Set 2

Skp structure after 10 ns simulation in vacuo (TXT 520 kb)

Supplementary Data Set 3

Skp structure after 7.5 ns simulation in explicit solvent (TXT 520 kb)

Supplementary Data Set 4

Starting model of 1:1 Skp–tOmpA complex used in MD simulations (TXT 738 kb)

Supplementary Data Set 5

Structure of 1:1 Skp–tOmpA complex after 10 ns simulation in vacuo (TXT 719 kb)

Supplementary Data Set 6

Starting model of 2:1 Skp–tBamA complex used in MD simulations (TXT 1491 kb)

Supplementary Data Set 7

Structure of 2:1 Skp–tBamA complex after 10 ns simulation in vacuo (TXT 1491 kb)

Supplementary Data Set 8

Structure of 1:1 Skp–tOmpA complex after 100 ns simulation in explicit solvent (TXT 719 kb)

Supplementary Data Set 9

Structure of 2:1 Skp–tBamA complex after 100 ns simulation in explicit solvent (TXT 1491 kb)

Molecular dynamics (MD) simulation of Skp in explicit solvent

A 100 ns MD simulation of Skp was performed to probe the dynamics of the Skp subunits in solution. The video (of the first 25 ns of the simulation) illustrates that all three Skp subunits undergo a transition from a ‘closed’ to an ‘open’ state in which the subunit splays out from the central axis. (MPG 4080 kb)

MD simulation of tOmpA in implicit solvent

A 3 ns simulation of tOmpA was performed to obtain a collapsed tOmpA structure for use in a subsequent simulation of a SkptOmpA complex in vacuo. The starting model for tOmpA in an extended conformation was generated in xleap. The video (of the first 0.6 ns of the simulation) shows tOmpA collapsing to an approximately globular structure. (MPG 1071 kb)

MD simulation of a Skp–tOmpA complex in vacuo

A 100 ns simulation of a model for the Skp–tOmpA complex was performed to gain insight into the structure of the Skp–tOmpA complex in the gas phase. The starting model was generated by placing a model of collapsed tOmpA within the cavity of Skp in an ‘open’ conformation. The video (of the first 1 ns of the simulation) illustrates the Skp subunits ‘clamping’ around the tOmpA substrate. The theoretical CCS value calculated for the Skp–tOmpA complex at the end of the simulation is in excellent agreement with that measured by IMS-MS (Supplementary Table 5). Skp is shown in secondary structure cartoon representation and tOmpA is shown as white spheres (vdw representation). (MPG 4838 kb)

MD simulation of 2:1 Skp–tBamA complex in vacuo

A 100 ns simulation of a model for the 2:1 Skp–tBamA complex was performed to gain insight into the structure of the 2:1 Skp–tBamA complex in the gas phase. The starting model was generated by placing a model of collapsed tBamA within the cavity formed by two copies of Skp in an ‘open’ conformation arranged in a side-by-side parallel orientation. The video (of the first 1 ns of the simulation) shows the subunits of both copies of Skp ‘clamping’ around the tBamA substrate. The theoretical CCS value calculated for the 2:1 Skp–tBamA complex at the end of the simulation is in excellent agreement with that measured by IMS-MS (Supplementary Table 5). The two copies of Skp are shown in secondary structure cartoon representation and tBamA is shown as white spheres (vdw representation). (MPG 8077 kb)

MD simulation of a Skp–tOmpA complex in explicit solvent

A 100 ns simulation of a model for the Skp–tOmpA complex was performed to gain insight into the structure of the Skp–tOmpA complex in solution. The starting model was generated by placing a model of collapsed tOmpA within the cavity of Skp in an ‘open’ conformation. The video (of the whole 100 ns of the simulation) illustrates the Skp subunits ‘clamping’ around the tOmpA substrate in solution, forming a stable complex. Skp is shown in secondary structure cartoon representation and tOmpA is shown as white spheres (vdw representation). (MPG 3100 kb)

MD simulation of 2:1 Skp–tBamA complex in explicit solvent

A 100 ns simulation of a model for the 2:1 Skp–tBamA complex was performed to gain insight into the structure of the 2:1 Skp–tBamA complex in solution. The starting model was generated by placing a model of collapsed tBamA within the cavity formed by two copies of Skp in an ‘open’ conformation arranged in a side-byside parallel orientation. The video (of the whole 100 ns of the simulation) shows the subunits of both copies of Skp ‘clamping’ around the tBamA substrate in solution, forming a stable complex. The two copies of Skp are shown in secondary structure cartoon representation and tBamA is shown as white spheres (vdw representation). (MPG 4035 kb)

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Schiffrin, B., Calabrese, A., Devine, P. et al. Skp is a multivalent chaperone of outer-membrane proteins. Nat Struct Mol Biol 23, 786–793 (2016). https://doi.org/10.1038/nsmb.3266

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