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. 2016 Jul 6;138(26):8143-55.
doi: 10.1021/jacs.6b03142. Epub 2016 Jun 23.

Solid-State NMR Investigation of the Conformation, Proton Conduction, and Hydration of the Influenza B Virus M2 Transmembrane Proton Channel

Jonathan K Williams  1 , Daniel Tietze  1 , Myungwoon Lee  1 , Jun Wang  2 , Mei Hong  1
Affiliations

Solid-State NMR Investigation of the Conformation, Proton Conduction, and Hydration of the Influenza B Virus M2 Transmembrane Proton Channel

Jonathan K Williams et al. J Am Chem Soc. .

Abstract

Together with the influenza A virus, influenza B virus causes seasonal flu epidemics. The M2 protein of influenza B (BM2) forms a tetrameric proton-conducting channel that is important for the virus lifecycle. BM2 shares little sequence homology with AM2, except for a conserved HxxxW motif in the transmembrane (TM) domain. Unlike AM2, no antiviral drugs have been developed to block the BM2 channel. To elucidate the proton-conduction mechanism of BM2 and to facilitate the development of BM2 inhibitors, we have employed solid-state NMR spectroscopy to investigate the conformation, dynamics, and hydration of the BM2 TM domain in lipid bilayers. BM2 adopts an α-helical conformation in lipid membranes. At physiological temperature and low pH, the proton-selective residue, His19, shows relatively narrow (15)N chemical exchange peaks for the imidazole nitrogens, indicating fast proton shuttling that interconverts cationic and neutral histidines. Importantly, pH-dependent (15)N chemical shifts indicate that His19 retains the neutral population to much lower pH than His37 in AM2, indicating larger acid-dissociation constants or lower pKa's. We attribute these dynamical and equilibrium differences to the presence of a second titratable histidine, His27, which may increase the proton-dissociation rate of His19. Two-dimensional (1)H-(13)C correlation spectra probing water (1)H polarization transfer to the peptide indicates that the BM2 channel becomes much more hydrated at low pH than at high pH, particularly at Ser12, indicating that the pore-facing serine residues in BM2 mediate proton relay to the proton-selective histidine.

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Figures

Figure 1
Figure 1
Comparison of the amino acid sequences (a) of the BM2 and AM2 transmembrane domains. Lowercase letters indicate heptad repeat, with a and d indicating pore-facing positions. The conserved HxxxW motif is underlined and the 13C, 15N-labeled residues are shown in red. (b) Four-helix bundle organization of the BM2 and AM2 transmembrane domains, with the pore-facing a and d residues indicated.
Figure 2
Figure 2
1D 13C CP-MAS spectra of BM2(1–33) as a function of pH, temperature, and membrane composition. (a) BM2 in the PC/PG/Chol membrane at pH 7.5. (b) BM2 in the VM+ membrane at pH 5.5. The VM+ membrane immobilizes the peptide while the PC/PG/Chol membrane promotes peptide motion at high temperature. (c–e) DNP-enhanced 13C spectra of BM2 at pH 5.5 in a partially deuterated VM+ membrane. (c) 13C CP spectrum with MW on. The sensitivity is 21–27 times that of the MW-off spectrum (not shown). (d) 15N-13C dipolar filtered 13C spectrum. (e) 13C DQF spectrum.
Figure 3
Figure 3
2D 13C-13C correlation spectra of site-specifically labeled BM2(1–33) in different membranes and at different pH. (a) PC/PG/Chol at pH 7.5. (b) PC/PG/Chol at pH 5.5. (c) PC/PG/Chol at pH 4.5. (d) VM+ membrane at pH 5.5. (e) VM+’ membrane at pH 5.5 with DNP. (f) VM+ membrane at pH 4.5.
Figure 4
Figure 4
His19 regions of the 2D 13C-13C correlation spectra of BM2(1–33) at different pH and in different lipid membranes. The spectra in (a–c) were measured at 243 K on PC/PG/Chol bound peptide. (a) pH 7.5, (b) pH 5.5, (c) pH 4.5. The spectra in (d–e) were measured at 263 K on VM+ bound peptide at (d) pH 5.5 and (e) pH 4.5. (f) DNP-enhanced spectrum of BM2(1–33) in the d31-VM+’ membrane at pH 5.5. Spectra (a–c) were measured on a 400 MHz spectrometer under 7 kHz MAS, while spectra (d–e) were measured on an 800 MHz spectrometer with 14.5 kHz MAS. The DNP spectrum (f) was measured on a 400 MHz/263 GHz spectrometer at 117 K under 9 kHz MAS. The DARR mixing times were 50 or 100 ms in these spectra.
Figure 5
Figure 5
2D 15N-13C correlation spectra of His19 in BM2(1–33). (a, b) BM2 in the VM+ membrane, measured on an 800 MHz spectrometer with 14.5 kHz MAS at 263 K. (a) pH 5.5. (b) pH 4.5. (c) BM2 at pH 5.5 in the d31-VM+’ membrane, measured on a 400 MHz/263 GHz DNP spectrometer under 9 kHz MAS at 117 K. Note the different neutral and cationic histidine distribution between high and low temperatures at pH 5.5, indicating temperature-induced pKa shifts.
Figure 6
Figure 6
Comparison of His19 and His37 conformational distribution in BM2 and AM2 from 2D 13C-13C correlation spectra. All peptides and proteins were bound to eukaryotic-mimetic lipid membranes, except for the pH 4.5 spectrum of BM2, which was bound to the PC/PG/Chol membrane. Spectra were measured at moderate low temperatures (243 – 273 K) where the peptides and proteins are immobilized. (a) BM2(1–33) in VM+ membranes at pH 5.5 and pH 4.5, and BM2(1–33) in the PC/PG/Chol membrane at pH 4.5. (b) AM2(21–97) in the VM+ membrane at pH 7.5 and pH 5.4. (c) AM2 TM peptide (residues 22–46) in the VM membrane at pH 7.0, 5.2 and 4.5. BM2 His19 exhibits the largest number of coexisting species, at pH 4.5. The fact that neutral tautomers persist to lower pH in BM2 indicates that His19 protonates with lower and more clustered pKa’s. The spectrum of the pH 4.5 VM+ bound BM2 resembles the spectrum of the PC/PG/Chol bound BM2 at pH 5.5 in Fig. 4b, indicating the influence of the negatively charged lipid on the protonation equilibria.
Figure 7
Figure 7
1D 15N CP spectra of BM2(1–33) as a function of pH, membrane composition and temperature. (a) Spectra of the VM+ bound BM2 from pH 4.0 to pH 6.5 at 243 K. (b) Spectra of PC/PG/Chol-bound BM2 at pH 5.5 and pH 7.5 at 243 K. (c) Comparison of high-temperature (303 – 308 K) 15N spectra of BM2 and AM2 at acidic pH. An 15N exchange peak is detected at ~213 ppm at pH 4.5 for BM2, at pH 5.2 for AM2-TM, and at pH 5.4 in the S31N mutant of AM2-TM. The exchange-averaged chemical shifts are the same in all M2 samples, but the exchange linewidths differ. The BM2 exchange peak is fit (gray line) to give a linewidth of 400±140 Hz.
Figure 8
Figure 8
pKa extraction of His19 in BM2(1–33). (a) NH to N intensity ratios as a function of pH. (b) Neutral-to-cationic histidine concentration ratios as a function of pH. The BM2 data (black) are compared with previously measured AM2 TM peptide data (orange) and AM2(21–97) data (blue). The neutral histidine concentration in BM2 is higher than that of AM2 at similar pH. The extracted pKa’s for BM2 are 6.1, 5.7, 4.5 and 4.2, whose average is lower than that of AM2 constructs. The average pKa’s of the three M2 samples are indicated as solid lines at the bottom. (c) Populations of charged tetrads of His19 in BM2 as a function of pH. The intercepts of adjacent population curves correspond to the pKa’s.
Figure 9
Figure 9
2D 1H-13C HETCOR spectra of membrane-bound BM2(1–33) to probe channel hydration. (a) Representative 2D spectrum of PC/PG/Chol bound peptide at pH 7.5, measured with a 1H spin diffusion mixing time of 50 ms. (b) Water cross sections of BM2(1–33) between 4 ms and 50 or 100 ms for the pH 7.5 and pH 5.5 PC/PG/Chol samples measured at 263 K. The S/S0 values are indicated for H19 and S12. (c) Water cross sections of the aliphatic region of the VM+ bound BM2 at pH 5.5 and pH 4.5, showing the large increase of the initial buildup of the S12-water cross peak intensity at lower pH. (d) Water cross section of the aliphatic region of VM-bound AM2 at pH 4.5, showing that the S31 S/S0 value is lower than that of S12 in BM2. (e) Initial buildup (S/S0 values) at high and low pH for PC/PG/Chol-bound BM2. (f) S/S0 values of VM+ bound BM2 at pH 5.5 and pH 4.5. (g) S/S0 values of the His19 sidechain in the two membranes. (h) S/S0 values of the Cα sites of AM2 residues and of the BM2 residues S12, A17 and G26 (shaded bars). S12 in BM2 shows the largest low-pH induced increase of the S/S0 value among all labeled residues of the two peptides. (i) Comparison of the solution NMR structure of BM2 (top, PDB: 2KIX) and AM2 (bottom, PDB: 2KQT) TM domains.
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