Abstract
The biogenesis of integral outer-membrane proteins (OMPs) in Gram-negative bacteria requires molecular chaperones that prevent the aggregation of OMP polypeptides in the aqueous periplasmic space. How these energy-independent chaperones interact with their substrates is not well understood. We have used high-resolution NMR spectroscopy to examine the conformation and dynamics of the Escherichia coli periplasmic chaperone Skp and two of its complexes with OMPs. The Skp trimer constitutes a flexible architectural scaffold that becomes more rigid upon substrate binding. The OMP substrates populate a dynamic conformational ensemble with structural interconversion rates on the submillisecond timescale. The global lifetime of the chaperone–substrate complex is seven orders of magnitude longer, emerging from the short local lifetimes by avidity. The dynamic state allows for energy-independent substrate release and provides a general paradigm for the conformation of OMP polypeptides bound to energy-independent chaperones.
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Acknowledgements
We thank R. Horst, P. Schanda, A. Gossert and G. Wider for discussions and D. Kahne (Harvard University) for the SurA plasmid. This work was supported by grants from the Swiss National Science Foundation (grant PP00P3_128419) and from the European Research Commission (FP7 contract MOMP 281764) to S.H. and by personal fellowships from the Novartis Foundation to B.M.B. and from the Werner-Siemens Foundation to C.W.
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B.M.B. and S.H. designed the study, analyzed the data, discussed the results and wrote the paper. B.M.B. and C.W. conducted the paramagnetic spin label experiments, and B.M.B. conducted all other experimental work.
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Supplementary Figure 1 Biophysical characterization of Omp–Skp complexes.
(a) Measurement of the molecular diffusion constant in aqueous solution with the 15N-filtered diffusion BPP-LED NMR experiment 1. Data for OmpX–Skp (green) and tOmpA–Skp (yellow). The logarithm of the signal intensity is plotted vs. the applied strength of the pulsed field gradients. The black lines are a linear fit to the data. These data are further analyzed in Supplementary Table 1. (b) Gel elution profiles of membrane-protein–chaperone complexes. Recorded at 8°C in assembly buffer on a Superdex S200 size exclusion column. The following protein concentrations were applied: Skp, 20 μM; Skp–tOmpA, 20 μM; Skp–OmpX, 20 μM; tOmpA, 4 μM; OmpX, 0.84 μM. The column void volume and the molecular weights of a standard calibration curve are indicated. These data are further analyzed in Supplementary Table 1.
Supplementary Figure 2 Sequence-specific NMR resonance assignments and backbone dynamics of Skp in its apo and holo forms.
(a–c) 2D [15N,1H]-TROSY fingerprint spectra of apo and holo forms of Skp. (a) Spectrum of 180 μM [U-2H,13C,15N]-apo Skp (black), (b) spectrum of 150 μM [U-2H,13C,15N]-Skp, with [U-2H]-OmpX bound (green), and (c) spectrum of 185 μM [U-2H,15N]-Skp with [U-2H]-tOmpA bound (yellow). The sequence specific resonance assignments obtained from 3D triple-resonance experiments are indicated. All spectra were recorded at 37°C. (d) Sequence-specific relaxation parameters of apo and holo Skp. The experimentally determined nuclear relaxation parameters R1(15N), R2(15N) and 15N{1H}-NOE and the resulting spectral densities J(0), J(wN) and J(0.87ωH) are plotted against the amino acid residue number of Skp. Color code (corresponding to panels a–c): apo Skp (black), holo-Skp with OmpX (green), and holo-Skp with tOmpA (yellow). All experiments were recorded at 37°C at a spectrometer 1H frequency of 700.2 MHz, corresponding to ⌉N = 440 ms-1 and 0.87⌉H = 3.8 ns-1. (e) Differences in the heteronuclear 15N{1H}-NOE between apo Skp and Skp–OmpX (left panel), and between apo Skp and Skp–tOmpA (right panel), plotted against the amino acid residue number of Skp. Negative values indicate a higher flexibility on the ps–ns timescale for apo Skp, positive values for the respective holo Skp form.
Supplementary Figure 3 Sequence-specific NMR resonance assignments and backbone dynamics of Omp substrates bound to Skp.
(a) 2D [15N,1H]-TROSY fingerprint spectrum of 270 μM of [U-2H,15N]-tOmpA bound to [U-2H]-Skp, recorded at 37°C. The sequence-specific resonance assignments obtained from 3D tripleresonance experiments are indicated. (b, c) Representative backbone assignment strips from 3D TROSYHNCACB experiments of (b) OmpX bound to Skp and (c) tOmpA bound to Skp. (d) Sequence-specific relaxation parameters R1(15N), R2(15N) and 15N{1H}-NOE for tOmpA bound to Skp (blue) and OmpX bound to Skp (purple), plotted against the amino acid residue numbers. Measurements were done at Omp–Skp concentrations of 270 μM and 320 μM, respectively, at 37°C and a spectrometer 1H frequency of 800.2 MHz. (e) Sequence-specific spectral densities J(0), J(⌉N) and J(0.87⌉H) of [U-2H,15N]-tOmpA bound to [U-2H]-Skp. At the spectrometer 1H frequency of 800.2 MHz, the frequencies are to ⌉N = 500 ms-1 and 0.87⌉H = 4.3 ns-1. Dashed lines indicate the average value of all amino acid residues in the respective polypeptide chains.
Supplementary Figure 4 Temperature-dependent line broadening of Skp-bound OmpX.
(a) 2D [15N,1H]-TROSY spectra of [U-2H,15N]-OmpX bound to [U-2H]-Skp at the temperatures of 37°C, 25°C, and 13°C. (b) ΔF are the free energies of transfer of the individual amino acids from an aqueous solution to its surface 2. Hydrophobicity corresponds to negative ΔF values. A linearly weighted 9-window average was applied to the raw data, with the edges contributing 50%. The red lines indicate the average value of + 0.8 standard deviations, the chosen threshold for the identification of the most hydrophilic segments. (c) Amino acid sequence of OmpX. The positions of the eight β-strands formed in natively folded OmpX are indicated. Green and orange bars indicate the observable and unobservable resonances at 13°C, respectively. Blue bars indicate the segments exhibiting the highest degree of hydrophilicity as identified in panel (b).
Supplementary Figure 5 Equilibration kinetics of the OmpX–Skp complex.
(a) Regions of 2D [15N,1H]-TROSY spectra of 90 μM of [U-2H,15N]-Skp with [U-2H]-OmpX (green) and upon the addition of 0.5 (blue) as well as 1.0 (cyan) equivalents of apo-[U-2H,15N]-Skp. The sequence-specific resonance assignments are indicated. The appearance of two distinct signals indicates slow exchange between the holo and the apo form of Skp, i.e. exchange rate constants < 1s-1. (b) Determination of the lifetime of the Skp–OmpX complex. 150 μM of unlabeled Skp–OmpX were mixed with 150 μM [U-2H,15N]-Skp at the start of the experiment. A series of 2D [15N,1H]-TROSY spectra (measurement time 230 min) were recorded. The volumes of distinct peaks of the apo and holo form were integrated (blue and red data points, respectively). Non-linear least squares fitting of a double exponential function (black line) yielded the global lifetime constant of the Skp–OmpX complex of 2.6 ± 0.9 h.
Supplementary Figure 6 NMR fingerprint spectra of tOmpA bound to different chaperones.
(a) Ribbon representation of the SecB crystal structure (PDB 1QYN, 3). 2D [15N,1H]-TROSY fingerprint spectrum of 150 μM of [U-2H,15N]-tOmpA bound to SecB (tetramer). (b) Ribbon representation of the crystal structure of the trigger factor monomer (PDB 1W26, purple 4). 2D [15N,1H]-TROSY fingerprint spectrum of 300 μM of [U-2H,15N]-tOmpA bound to trigger factor (dimer). (c) Ribbon representation of the SurA crystal structure (PDB 1M5Y orange 5). 2D [15N,1H]-TROSY fingerprint spectrum of 250 μM of [U-2H,15N]-tOmpA bound to SurA (dimer). (d) Ribbon representation of the crystal structure of Skp (PDB 1SG2, blue 6). 2D [15N,1H]-TROSY fingerprint spectrum of 270 μM of [U-2H,15N]-tOmpA bound to [U-2H]-Skp. All spectra were recorded at 37°C in NMR buffer.
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Burmann, B., Wang, C. & Hiller, S. Conformation and dynamics of the periplasmic membrane-protein–chaperone complexes OmpX–Skp and tOmpA–Skp. Nat Struct Mol Biol 20, 1265–1272 (2013). https://doi.org/10.1038/nsmb.2677
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DOI: https://doi.org/10.1038/nsmb.2677
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