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Atomic structures of closed and open influenza B M2 proton channel reveal the conduction mechanism

Abstract

The influenza B M2 (BM2) proton channel is activated by acidic pH to mediate virus uncoating. Unlike influenza A M2 (AM2), which conducts protons with strong inward rectification, BM2 conducts protons both inward and outward. Here we report 1.4- and 1.5-Å solid-state NMR structures of the transmembrane domain of the closed and open BM2 channels in a phospholipid environment. Upon activation, the transmembrane helices increase the tilt angle by 6° and the average pore diameter enlarges by 2.1 Å. BM2 thus undergoes a scissor motion for activation, which differs from the alternating-access motion of AM2. These results indicate that asymmetric proton conduction requires a backbone hinge motion, whereas bidirectional conduction is achieved by a symmetric scissor motion. The proton-selective histidine and gating tryptophan in the open BM2 reorient on the microsecond timescale, similar to AM2, indicating that side chain dynamics are the essential driver of proton shuttling.

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Fig. 1: Two-dimensional correlation solid-state NMR spectra indicate an α-helical TM domain for membrane-bound BM2.
Fig. 2: Determination of the helix orientation of BM2 in the closed (high pH) and open (low pH) states.
Fig. 3: Determination of interhelical distances of membrane-bound BM2 using 13C-19F REDOR.
Fig. 4: Atomic structures of closed and open BM2 channels in lipid membranes.
Fig. 5: Water accessibilities and side chain motions of BM2 in the high-pH (closed) and low-pH (open) states.

Data availability

NMR chemical shifts and the torsion angle, distance and orientation restraints for high-pH and low-pH BM2 have been deposited in the Biological Magnetic Resonance Bank (BMRB) under ID nos. 30645 and 30646. The structural coordinates for the high-pH closed and low-pH open BM2 have been deposited in the Protein Data Bank under accession codes 6PVR and 6PVT.

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Acknowledgements

This research is funded by National Institutes of Health (NIH) grant no. GM088204 to M.H. We thank B. Kwon for help with protein purification and M.D. Gelenter and A.J. Dregni for discussions about structure calculation. This study made use of NMRbox: National Center for Biomolecular NMR Data Processing and Analysis, a Biomedical Technology Research Resource (BTRR), which is supported by NIH grant no. P41GM111135 (NIGMS).

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Contributions

V.S.M. and A.R.L. carried out BM2 cloning. V.S.M. expressed and purified BM2. A.R.L. and B.L.P. synthesized and purified fluorinated BM2 peptide. V.S.M. conducted most of the SSNMR experiments, assigned and analyzed the spectra and calculated the structures. A.A.S. contributed to the 13C-19F distance experiments and data analysis. M.H. designed the experiments and supervised data analysis. M.H. and V.S.M. wrote the paper, with input from other authors. All authors discussed the results of the study.

Corresponding author

Correspondence to Mei Hong.

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The authors declare no competing interests.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Purification and characterization of BM2(1–51).

a, Amino acid sequences of the TM domain of AM2 and BM2. The conserved proton-selective histidine and the gating tryptophan are shown in red. The other pore-lining heptad a and d residues are polar in BM2 and hydrophobic in AM2 (blue). b, SDS-PAGE gel showing Ni2+-affinity purification of SUMO-BM2. The flow through contains all soluble cellular proteins with low affinity for Ni2+. The column was washed with 50 mM imidazole, and SUMO-BM2 (18 kDa band) was eluted in two fractions at >90% purity with 300 mM imidazole. c, Analytical reverse-phase HPLC chromatogram of BM2 before (black) and after (red) protease cleavage of the SUMO tag to give native BM2 at an elution time of 11.2 min. d, MALDI mass spectrum of purified BM2(1-51), showing excellent agreement between the observed mass and the theoretical mass. e, Circular dichroism spectrum of BM2 in 0.5% n-dodecylphosphocholine solution at pH 7.5. Spectral deconvolution indicates 60% α-helicity and 40% disordered or turn structures. f, LC-MS total ion chromatogram of purified 4-19F-Phe5, 4-19F-Phe20 labeled synthetic BM2(1-51), showing excellent purity. g, Deconvolution of extracted ion chromatogram of purified 4-19F-Phe5, 4-19F-Phe20 BM2. The measured molecular weight is in excellent agreement with the expected molecular weight.

Extended Data Fig. 2 Resonance assignment and inter-residue correlations of membrane-bound BM2 at pH 4.5.

a, Representative strips of the NCACX (orange) and NCOCX (blue) regions of the 3D NCC spectrum to obtain sequential resonance assignment. The spectrum was measured at Tsample = 280 K. b, Representative F2-F3 planes of the 3D CCC spectrum, showing various inter-residue correlations (assigned in red) that restrain the structure. The spectrum was measured using spin diffusion mixing times of 41 ms and 274 ms, at Tsample = 280 K. c, 2D 13C-13C TOCSY spectrum with 7.7 ms mixing, collected at Tsample = 290 K. Residues 43–51 are dynamic and exhibit chemical shifts indicative of random coil conformation. d, 1D 13C cross-polarization (CP) spectrum preferentially detects immobilized residues while the 13C INEPT spectrum preferentially detects highly dynamic residues. These 1D spectra were measured at Tsample = 280 K.

Extended Data Fig. 3 Resonance assignment and inter-residue correlations of membrane-bound BM2 at pH 7.5.

a, 2D 13C-13C correlation spectrum with 55 ms CORD spin diffusion, measured at Tsample = 280 K. b, Representative F2-F3 strips from the 3D CCC spectrum, showing various inter-residue correlations (assigned in red) that restrain the structure. The spectrum was measured using spin diffusion mixing times of 41 ms and 274 ms, at Tsample = 280 K. c, 2D 13C-13C TOCSY spectrum with 7.7 ms mixing, collected at Tsample = 290 K. Residues 43–51 are dynamic and exhibit chemical shifts indicative of random coil conformation. d, 13C CP spectrum preferentially detects immobilized residues while the 13C INEPT spectrum preferentially detects highly dynamic residues. These 1D spectra were measured at Tsample = 280 K.

Extended Data Fig. 4 Secondary structure of BM2 in the closed (high pH) and open (low pH) states.

a, Cα (black) and Cβ (magenta) secondary chemical shifts at pH 7.5 and pH 4.5. b, Chemical-shift derived (ϕ, ψ) torsion angles at pH 7.5 (black) and pH 4.5 (orange). At both pH, the TM domain is α-helical while the cytoplasmic tail is mostly disordered. In addition, a short β-strand segment is present at low pH. c, Helical wheel representations of residues 48-63 in AM2 and the corresponding residues 29-44 in BM2. Hydrophobic residues are colored green, polar residues black, positively charged residues blue, and negatively charged residues red. AM2 has a separate hydrophobic face and a hydrophilic face, indicative of an amphipathic helix, while BM2 has alternating polar and non-polar residues, consistent with a β-strand conformation. d, Static 31P spectra of BM2-containing POPE membrane at high and low pH and POPC/POPG membranes at low pH, all measured at a sample temperature of 303 K. At high pH the POPE membrane consists of ~65% bilayer and ~35% hexagonal phase. At low pH BM2 converts most of the POPE membrane to the hexagonal phase, but retains the lamellar form for the POPC/POPG membrane. Green dashed line is a superposition of 35% of the pH 4.5 POPE spectrum and 65% of the pH 4.5 POPC: POPG spectrum.

Extended Data Fig. 5 BM2 has similar conformations in POPE and POPC:POPG bilayers.

a, 2D 13C-13C CORD spectra of BM2 in the two lipid membranes at low pH. b, 2D 15N-13C correlation spectra of BM2 in the two lipid membranes at low pH. The POPE sample was measured at Tsample = 290 K for the 2D NC spectrum and 280 K for the 2D CC spectrum, while the POPC: POPG sample was measured at Tsample = 270 K to account for the lower phase transition temperature of this membrane. The lipid bilayers of both samples were in the gel phase, as assessed by 1H spectra of the sample. Both spectra were measured under 14 kHz MAS on an 800 MHz spectrometer. c, Chemical shift differences between the POPE and POPC:POPG samples at low pH. Residues in the α-helical TM domain and the β-strand do not show significant chemical shift differences.

Extended Data Fig. 6 Measurement of BM2 helix orientation using rotationally averaged 15N-1H dipolar couplings.

a,b, N-H DIPSHIFT data of the tripeptide formyl-MLF, measured at Tsample = 315 K using (a) 15N detection and (b) 13C detection. The dipolar-doubled version of DIPSHIFT is used in these experiments. The 15N-detected DIPSHIFT data were analyzed using the total intensities from the centerband and sidebands. The 13C-detected N-H couplings used a 15N-13C TEDOR mixing time of 2.11 ms. The 13C-detected N-H couplings are 0.9 times the 15N-detected values, indicating incomplete powder averaging. This scaling factor was included in determining the BM2 orientation from 13C-detected N-H dipolar couplings. c, Calculated 15N-1H dipolar waves as a function of the helix tilt angle. An 18-residue ideal α-helix with (ϕ, ψ) angles of (-65˚, -40˚) were tilted from an external axis by 0°–30°. The 15N-1H dipolar couplings show the expected sinusoidal oscillations with a periodicity of 3.6 residues. The amplitude and offset of the dipolar wave indicate the helix tilt angle. d, Reduced χ2 values of the measured and simulated 15N-1H dipolar couplings of membrane-bound BM2 at high and low pH. The minimum χ2 value is found at a tilt angle of 14˚ for high-pH BM2 and 20˚ for low-pH BM2. The ±2˚ uncertainty represents one standard deviation.

Extended Data Fig. 7 13C-19F REDOR data for measuring interhelical distances of BM2 at high pH (black curves and filled symbols) and low pH (orange curves and open symbols).

The high pH data were measured at a sample temperature (Tsample) of 273 K, while the low pH data were measured at 261 K. Additional high-pH data measured at Tsample = 261 K (red symbols in some of the panels) are indistinguishable from 273 K data, confirming that the protein is immobilized at both temperatures. a, N-terminal residues that are dephased by 4F-Phe5. b, C-terminal residues whose dephasing is attributed to 4F-Phe20. All sites show less dephasing for the low-pH sample than the high-pH sample, indicating longer distances for the open channel. P4 has negligible dephasing at low pH. c, Representative χ2 as a function of 13C-19F distance, showing the extraction of the best-fit distances and uncertainties. d, Aromatic region of representative 13C-19F REDOR spectra of BM2 at high pH. The difference spectrum (∆S) shows no dephasing for the 119-ppm W23 Cε3/ζ3/η2 peak (blue dashed line), indicating that 4F-Phe20 of the neighboring helix is far from these indole carbons. e, This is consistent with a W23 rotamer of t90 (χ1 = −125°, χ2 = 98°) but inconsistent with the mt rotamer (χ1 = -80°, χ2 = -177°).

Extended Data Fig. 8 HxxxW motif rotamers and comparison of the closed BM2 structures in lipid bilayers versus detergent micelles.

a, Structural ensembles of H19 and W23 in the conserved HxxxW conduction motif at high pH (left) and low pH (right). The H19 χ1 is trans but the χ2 is not constrained well by experimental data. W23 predominantly adopts the t90 rotamer in both closed and open structural ensembles. b,c, Comparison of the high-pH BM2 TM structure in lipid bilayers versus in detergent micelles. b, Solid-state NMR structure determined here in POPE membranes. c, Solution NMR structure determined in DHPC micelles1.

Extended Data Fig. 9 Hydration of membrane-bound BM2.

a, Aliphatic region of the 13C spectra measured with 100 ms (black) and 2 ms (red) 1H polarization transfer from water to the protein, measured at Tsample = 273 K. The low-pH protein shows higher intensities, indicating higher water accessibility. b, Aromatic region of the 13C spectra also show significantly higher water-transferred intensities at low pH than high pH. c, Water-to-protein polarization transfer curves for various residues. The buildup rates are faster at low pH (orange) than at high pH (black). d, 1D 15N CP spectra of the H19 and H27 side chains of BM2 at high and low pH, measured at Tsample = 280 K. The imidazole 15N signals are shifted 8–9 ppm downfield at low pH compared to high pH, indicating increased protonation of the histidines. e, Control 2D 13C-13C correlation spectrum, measured using a 1H-1H spin diffusion time of 100 ms to allow water magnetization to equilibrate with the protein. The spectra were measured at Tsample = 273 K.

Extended Data Fig. 10 Pulse sequences of key 2D and 3D correlation experiments used for determining the structures of closed and open BM2 channels.

a, 3D CCC experiment. The first 13C spin diffusion period is short to obtain intra-residue correlations while the second is long to obtain inter-residue cross peaks. b, Water-edited 2D CC experiment. A selective 90˚ pulse excites the water 1H magnetization, a 1H T2 filter removes the rigid protein magnetization, then the water magnetization is transferred to the protein. Filled and open rectangles indicate 90° and 180° pulses, respectively. c, 3D NCC experiment involving an out-and-back 15N-13C TEDOR period followed by 13C spin diffusion. The experiment simultaneously detects NCACX and NCOCX correlations. d, Frequency-selective 13C-19F REDOR for distance measurements. e, 3D NC-resolved N-H dipolar-doubled DIPSHIFT experiment for measuring helix orientations.

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Mandala, V.S., Loftis, A.R., Shcherbakov, A.A. et al. Atomic structures of closed and open influenza B M2 proton channel reveal the conduction mechanism. Nat Struct Mol Biol 27, 160–167 (2020). https://doi.org/10.1038/s41594-019-0371-2

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