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References
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B.P.O. performed protein expression, sample preparation, NMR experiments and data analysis, SEC–MALS experiments and analysis and helped write the paper; J.H.B. performed sample preparation, SEC–MALS experiments and analysis and helped write the paper; M.H. performed protein expression, sample preparation and SEC–MALS experiments; J.K.C. performed NMR experiments and data analysis; C.C., F.D. and N.H. performed molecular dynamics simulations; J.R.S. performed NMR experiments and reanalyzed NOESY spectra; N.Z. performed protein expression and sample preparation; J.R.S., N.Z., C.C. and F.D. conceived the study; and J.R.S. and N.Z. wrote the paper.
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Extended data figures and tables
Extended Data Fig. 1 Molecular dynamics of p7.
a, b, Insertion of the proposed hexameric p7 structure2 into lipid bilayers. MemProtMD6 prediction for the hexamer insertion into a hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayer (a). The p7 structure2 after insertion into a hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayer and simulated for 60 ns (b). Severe deformations and thinning defects of the bilayer can be seen, resulting in a large number of water molecules within the hydrophobic region of the bilayer. Water is shown as van der Waals spheres for oxygen (red) and hydrogen (white). For DPPC and POPC lipids, phosphorus and choline nitrogen positions are indicated with orange spheres and green spheres, respectively. In b, p7 α-helices and 310-helices are shown in magenta and blue, respectively. c–e, Simulations of monomeric p7 in 300 mM DPC at a protein to detergent ratio of 1:250. Independent 100-ns simulations of the horseshoe-like subunit conformation of the proposed hexameric p7 structure (c, d). At the end of the simulation, ~170 (c) and ~120 DPC (d) molecules, were observed bound to the protein. A hairpin conformation of p7 was simulated for 100 ns, at the end of which ~100 DPC molecules were observed bound to the protein (e). In c–e, p7 α-helices and 310-helices are shown in magenta and blue, respectively; the geometric centre of the DPC headgroup is indicated by an orange sphere, and the DPC hydrocarbon chain as yellow sticks. Only those DPC molecules bound to p7 are shown. The simulations in b–e were performed with the CHARMM36 all-atom force field using the protocol described19.
Extended Data Fig. 2 NMR spectroscopy of p7 in DPC.
a, Comparison of NMR spectra of p7, prepared as described2, with previously published spectra. Left, 2D 1H–15N-TROSY spectrum of a sample of p7 in DPC produced in the present study (red cross peaks), recorded at 30 °C with a 1H frequency of 600 MHz, overlaid with the spectrum published by OuYang et al.2 (blue cross peaks). Right, our p7 spectrum (red cross peaks) overlaid with another published spectrum8 (blue cross-peaks) to illustrate sample-to-sample variation. In both cases, the spectra were overlaid manually. b, 1H–15N HSQC of p7 in DPC at 37 °C. The spectrum was recorded at 600 MHz (1H) on a Bruker spectrometer equipped with a CryoProbe. c, Temperature sensitivity of the spectrum of p7 in DPC. Spectra were recorded at three temperatures: 30 °C (blue), 33.5 °C (green) and 37 °C (red). The spectra have not been corrected for the temperature dependence of the 2H-lock frequency. d, NMR spectra of p7 under SEC–MALS conditions. Spectra were recorded of 5 µM p7 with 50 mM DPC (detergent-to-protein ratio of 10,000) to match conditions at the SEC–MALS peak summit for a sample prepared as described2 (violet) (see Fig. 1a and Extended Data Table 1), or as described by Dev et al.8, with pre-gel filtration in 3 mM DPC buffer containing 100 mM NaCl (orange) (see Extended Data Fig. 3a). Before recording the NMR data, the sample in 3 mM DPC + 100 mM NaCl was dialysed against buffer (25 mM MES at pH 6.5, 3 mM DPC, and 1 mM DSS with 5% D2O) overnight to remove salt, concentrated, and then diluted with NMR buffer (25 mM MES at pH 6.5, 50 mM DPC, and 1 mM DSS with 5% D2O) to match the concentrations at the peak summit of the SEC–MALS elution (5 µM p7 and 50 mM DPC). Band-selective excitation short-transient transverse relaxation-optimized spectroscopy (BEST-TROSY) NMR experiments were recorded over ~20 h each on a 750-MHz spectrometer equipped with a CryoProbe to obtain sufficient signal-to-noise ratios for the dilute samples and overlaid with the published TROSY spectrum (supplementary figure 2a in OuYang et al.2). e, Backbone amide chemical-shift differences between a sample of p7 in DPC without and with 5 mM rimantadine. Data analysis was carried out on the spectra of OuYang et al.2. Chemical-shift differences were calculated as indicated on the vertical axis for the backbone amide resonances in the 15N–2H-labelled mixed-label sample and the 15N–2H-labelled sample containing 5 mM rimantadine. Residues in pink were previously identified2 as forming the rimantadine-binding pocket (see figure 3c in ref. 2). All reported concentrations are for monomeric protein.
Extended Data Fig. 3 SEC–MALS of p7 and detergent alone.
a, P7 in 50 mM DPC prepared as described by Dev. et al.8. The sample was run over two Superdex 200 5/150 columns in series in order to resolve the protein and micelle complex. The calculated masses were 7.8 ± 1.1 and 24.7 ± 0.6 kDa for protein and the associated detergent, respectively. The DPC concentration and maximum p7 monomeric concentration eluted (summit of peak) are indicated above the graph. b–e, SEC–MALS of p7 in 80 mM and 10 mM SDS. The SDS concentration and the maximum concentration of eluted monomeric p7 (summit of peak) are indicated above each graph. p7 is monomeric in SDS, as shown by its mobility on SDS–PAGE2 (and our data, not shown). SEC–MALS molar mass analysis indicates, unambiguously, and in all cases, a monomeric state for p7 in SDS. The analysis also confirms the trend seen with DPC (Fig. 1 and a above), in that the amount of detergent associated with the protein is markedly and consistently higher for the samples at the lower detergent concentration (10 mM SDS) compared with samples in higher detergent concentration (80 mM SDS) (see data summary in Extended Data Table 1). Samples were run through two Superdex 200 5/150 columns connected in series for increased resolution. Sample injection volumes were 30 μl. Running buffer contained the indicated SDS concentrations and 10 mM sodium phosphate at pH 7.2. f–i, SEC–MALS of DPC and SDS micelles in the absence of protein. The detergent, its concentration in the injected sample and running buffers, and the chromatography columns used are indicated above each graph. As in b–e, SDS samples were run over two Superdex 200 5/150 in series to increase resolution. The A280 nm in samples without protein is negligible. Running buffers are the same as those used in experiments with protein (Fig. 1 and a–e), and the injected samples have higher detergent concentration to enable detection above the baseline. In i, the running buffer contained 30 mM SDS because the smaller size of micelles in 80 mM SDS in the absence of protein resulted in low signal-to-noise scattering. Molar masses (Det, orange line, right axis) increase slightly (by ~3 kDa for DPC and ~10 kDa for SDS) at concentrations close to the critical micelle concentration. Right axes: molar masses calculated at each point for protein (Prot; if protein is present), associated detergent (Det), and the detergent and protein complex (Det + Prot; if protein is present). Left axes: UV, A280 nm; LS, light scattering; RI, refractive index. Detector signals are scaled to enable comparison. In a, the inset shows the detector signals normalized at the p7 peak. Extended Data Tables 1 and 2 show a summary of experimental conditions and mass calculations. The reported masses denote the value at the peak summit and the error is taken as the maximum difference from this value across the elution volume for which the molar mass is plotted. For samples with p7, negative and positive scattering and refractive-index peaks following the protein peaks are the result of distortions of the baseline upon sample injection that causes disequilibrium of detergent micelles in the running buffer20.
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Oestringer, B.P., Bolivar, J.H., Hensen, M. et al. Re-evaluating the p7 viroporin structure. Nature 562, E8–E18 (2018). https://doi.org/10.1038/s41586-018-0561-9
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DOI: https://doi.org/10.1038/s41586-018-0561-9
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