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. 2023 Aug:299:107028.
doi: 10.1016/j.bpc.2023.107028. Epub 2023 May 13.

Lipid acyl chain protrusion induced by the influenza virus hemagglutinin fusion peptide detected by NMR paramagnetic relaxation enhancement

Yijin Zhang  1 , Ujjayini Ghosh  1 , Li Xie  1 , Daniel Holmes  1 , Kathryn G Severin  1 , David P Weliky  2
Affiliations

Lipid acyl chain protrusion induced by the influenza virus hemagglutinin fusion peptide detected by NMR paramagnetic relaxation enhancement

Yijin Zhang et al. Biophys Chem. 2023 Aug.

Abstract

The glycoprotein spikes of membrane-enveloped viruses include a subunit that catalyzes fusion (joining) of the viral and target cell membranes. For influenza virus, this is subunit 2 of hemagglutinin which has a ∼ 20-residue N-terminal fusion peptide (Fp) region that binds target membrane. An outstanding question is whether there are associated membrane changes important for fusion. Several computational studies have found increased "protrusion" of lipid acyl chains near Fp, i.e. one or more chain carbons are closer to the aqueous region than the headgroup phosphorus. Protrusion may accelerate initial joining of outer leaflets of the two membranes into a stalk intermediate. In this study, higher protrusion probability in membrane with vs. without Fp is convincingly detected by larger Mn2+-associated increases in chain 13C NMR transverse relaxation rates (Γ2's). Data analysis provides a ratio Γ2,neighbor/Γ2,distant for lipids neighboring vs. more distant from the Fp. The calculated ratio depends on the number of Fp-neighboring lipids and the experimentally-derived range of 4 to 24 matches the range of increased protrusion probabilities from different simulations. For samples either with or without Fp, the Γ2 values are well-fitted by an exponential decay as the 13C site moves closer to the chain terminus. The decays correlate with free-energy of protrusion proportional to the number of protruded -CH2 groups, with free energy per -CH2 of ∼0.25 kBT. The NMR data support one major fusion role of the Fp to be much greater protrusion of lipid chains, with highest protrusion probability for chain regions closest to the headgroups.

Keywords: Fusion peptide; Hemagglutinin; Influenza; NMR; PRE; Protrusion.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.
Pictorial representation of a common membrane fusion model that includes (a) initial close apposition of the viral and target membranes; (b) stalk formed from the outer leaflets of the two membranes; (c) hemifusion diaphragm that is contiguous with the inner leaflets of the two membranes; and (d) pore formation. The estimates of the free energy barriers for membrane apposition and for transformation between membrane intermediates are from computational studies of uncatalyzed fusion. The figure doesn’t show the final step of pore expansion that precedes full contents mixing. The different colors of the headgroups are meant to visually enhance the changes in membrane topology during fusion but don’t describe the locations of specific lipids during fusion. During the ~20 s estimated lifetime of a membrane intermediate structure in viral fusion, a lipid molecule could diffuse over ~1010 Å2 leaflet area.
Figure 2.
Figure 2.
Representative picture of lipid acyl chain protrusion near a Fp. A set of 128 POPC and 32 POPG lipids in a pre-assembled bilayer were energy-minimized in a water box using the CHARMM/Membrane Builder/Bilayer Builder/Membrane Only System molecular dynamics program. A membrane cross-section is displayed with lipid acyl chains in light green. A representative protruded chain in magenta is next to a Fp backbone in turquoise and near a Mn2+ in purple that is bound to a lipid headgroup. The picture shows a protruded palmitoyl chain and a Fp with semi-closed structure with marked N- and C- termini. There is increased protrusion in simulations for both palmitoyl and oleoyl chains and for lipids next to a variety of Fp structures. In addition, Fp in simulations is observed with both interfacial and transmembrane locations and protruded lipids exhibit a variety of geometries relative to the neighboring Fp.
Figure 3.
Figure 3.
Chemical structures of POPC and POPG lipids with site numbering of the acyl chains with prime (‘) for the palmitoyl chain and no prime for the oleoyl chain.
Figure 4.
Figure 4.
CP-MAS/Hahn-echo sequence displayed as rf field vs. time. Typical parameters included 1H transmitter at 3.5 ppm, 13C transmitter at 100.0 ppm, 8.0 kHz MAS frequency, 2.5 μs 1H π/2 pulse, 1.4 ms CP contact time, an array of τ between ~2 and ~40 ms with τ/2 an integral number of rotor periods, 10.0 μs 13C π pulse, SPINAL-64 1H decoupling during the Hahn-echo and acquisition periods, 13C acquisition with 25 μs dwell time and 1400 complex points, 1 s recycle delay, and sum of 8 or 16 K acquisitions using the phase cycle described in the text. Typical 1H rf fields are: CP, linear ramp between 44 and 60 kHz; and decoupling, 50 kHz. The typical 13C CP rf field is 42 kHz.
Figure 5.
Figure 5.
13C NMR spectra of samples containing (a) Lipid, (b) Lipid + Mn2+, (c) Lipid + Fp, and (d) Lipid + Fp + Mn2+, with 0.5% Mn2+ and 3% Fp that are calculated as (mole Mn2+ or Fp)/(mole lipid) × 100. There is POPC:POPG (4:1) lipid composition. The data were acquired after CP without the Hahn echo. The vertical scales of the four spectra have been adjusted so that the “*” peaks at 30 ppm have the same height. Assignments are displayed for the lipid acyl chain peaks use the Fig. 2 carbon numbering with prime (‘) for the palmitoyl chain and no prime for the oleoyl chain. There isn’t resolution of individual peaks for POPC vs. POPG. There are resolved peaks for sites with distinctive bonding environments, but with superposition of signals from at least two sites. Peaks are assigned for: (1) the 2,2’ and 3,3’ sites that are one and two bonds from the carbonyl groups; (2) the 9,10 C=C and 8,11 C=C adjacent sites of the oleoyl chain; (3) the 18,16’ -CH3 sites; and (4) the 17,15’ and 16,14’ sites that are one and two bonds from the -CH3 groups. The * peak is a superposition of signals from the 4–7, 12–15, and 4’−13’ sites. The carbonyl peak and headgroup peak regions are also noted.
Figure 6.
Figure 6.
The 2,2’ spectral signals vs. Δτ (increment in dephasing time) of samples containing (a) Lipid, (b) Lipid + Mn2+, (c) Lipid + Fp, and (d) Lipid + Fp + Mn2+, with 0.5% Mn2+ and 3% Fp that are based on (mole Mn2+ or Fp)/(mole lipid) × 100. Spectra were acquired with the CP-Hahn echo sequence. The vertical scales of the spectra of each sample were adjusted so that the Δτ = 0 spectral peaks have the same height. For these top spectra, τ = 1.25 ms for (a) and 2.00 ms for (b-d).
Figure 7.
Figure 7.
Integrated peak intensities vs. dephasing time (τ) and best-fit single exponential decays for the Lipid, Lipid + Mn2+, Lipid + Fp, and Lipid + Fp + Mn2+ samples. Data and fittings are displayed for the (a) 2,2’; (b) 3,3’; (c) 9,10; and (d) * peaks. The * peak is a superposition of signals from the 4–7, 12–15, and 4’−13’ sites. The peak intensities vs. τ were fitted to A × exp(−R2 × τ) with A and R2 as fitting parameters. The displayed intensities have been divided by A so that the best-fit intensity = 1 for all peaks when τ = 0. The best-fit R2’s and their uncertainties are presented in Table 2. The uncertainties in the peak intensities were calculated as the RMSD’s of ten different integrals in noise regions of the spectra. For the two-site peaks, these uncertainties were typically between 10−3 and 10−2 and less than the dimension of the points in the plots.
Figure 8.
Figure 8.
Bar plot of the Γ2’s, i.e. the differences between the R2’s for samples with vs. without Mn2+. The Γ2’s are displayed for several peaks. Each peak is due to signals from two 13C sites in the acyl chains. The Γ2’s are shown for samples without vs. with Fp. The Γ2’s and their uncertainties are presented numerically in Table 2.
Figure 9.
Figure 9.
Plots and exponential decay fittings of Γ2 vs. average number (n) of protruded -CH2. For each peak corresponding to signals from two -CH2 sites that are numbered x and y (Figs. 3 and 5), this average number is calculated as [(x+y)/2] – 1, e.g. 2 for the 3,3’ peak and 14 for the 16,14’ peak. Data are displayed for samples without and with Fp. Separate fittings are done for each sample type using Γ2 (n) = Γ2 (0) × exp(−n × κ) with Γ2 (0) and κ as fitting parameters and κ = ΔGprot/kBT. Best-fit values with uncertainties in parentheses are: (1) without Fp, Γ2 (0) = 27.9(1.8) s−1 and κ = 0.249(18); and (2) with Fp, Γ2 (0) = 47.3(2.6) s−1 and κ = 0.266(16).
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