NMR determination of protein partitioning into membrane domains with different curvatures and application to the influenza M2 peptide

Abstract

The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use (31)P and (13)C solid-state NMR to examine M2-induced membrane curvature. M2(22-46), which includes only the transmembrane (TM) helix, and M2(21-61), which contains an additional amphipathic helix, are studied. (31)P chemical shift lineshapes indicate that M2(21-61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22-46) has minimal effect. The lamellar and isotropic domains have distinct (31)P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. (31)P- and (13)C-detected (1)H T(2) relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21-61) preferentially binds to the high-curvature domain. (31)P linewidths indicate that the isotropic vesicles induced by M2(21-61) are 10-35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides.

Figure 1

Static ³¹P spectra of DMPC (a–c) and VM (d–i) membranes in the absence and presence of M2 at 303 K. (a, d, and g) Protein-free lipid bilayers. (b, e, and h) M2TM-containing membranes. (c, f, and i) M2(21–61)-containing membranes. The DMPC spectra (a–c) and the VM spectra (d–f) were measured on samples with Amt, whereas the VM spectra (g, h, and i) were measured on samples without Amt.

Figure 2

Static (black) and MAS (red) ³¹P spectra of lipid membranes without and with M2. Inset expands the MAS spectra. (a) M2(21–61)-DMPC membranes at 303 K. (b) DMPC isotropic vesicles at 298 K. (c) M2TM-VM membranes at 303 K. (d) M2(21–61)-VM membrane at 303 K. An isotropic peak at about +2 ppm is observed in a and d.

Figure 3

³¹P- and ¹³C-detected ¹H T₂ relaxation of M2-containing membranes at 298 K under 7 kHz MAS. Red: ³¹P-detected ¹H T₂ decay of lamellar lipids. Green: ³¹P-detected ¹H T₂ decay of isotropic lipids. Blue: ¹³C-detected ¹H T₂ decay of the peptide. Solid triangles: S31 Cα/Cβ. Open triangles: G34 Cα. (a) M2(21–61)-DMPC membranes with ¹H spin diffusion times of 25 ms for ³¹P and 49 ms for ¹³C. (b) M2(22–46)-VM membranes. Solid circles: −0.37 ppm lamellar ³¹P peak. Open circles: −1.0 ppm lamellar ³¹P peak. ¹H spin diffusion mixing times: 100 ms for ³¹P and 25 ms for ¹³C. (c) M2(21–61)-VM membranes with a 25 ms ¹H spin diffusion mixing time.

Figure 4

³¹P-detected ¹H T₂ relaxation decays without (black) and with (red and green) M2 at 298 K. (a) DMPC bilayers with and without M2(21–61). (b) VM membranes with and without M2(22–46). (c) VM membranes with and without M2(21–61).

Figure 5

¹H-¹³C and ¹H-³¹P 2D HETCOR spectra of M2(21−61)-DMPC membranes. (a) ¹H-¹³C HETCOR spectrum without ¹H spin diffusion. The S31 Cα/Cβ¹H cross section is shown in d. (b) ¹H-¹³C HETCOR spectrum with 200 μs ¹H spin diffusion. The S31 cross section is shown in e. (c) ¹H-³¹P HETCOR spectrum with 250 μs ¹H spin diffusion and 3 ms Lee-Goldburg cross-polarization. The ¹H cross sections at +1.9 ppm and −1.1 ppm are shown in f and g, respectively. All spectra were measured at 297 K with ¹H homonuclear decoupling under 7.5 kHz MAS.

Figure 6

Models of the effects of M2 peptides on membrane curvature. (a) M2TM does not cause curvature to the VM membrane and adopts a conformation competent to bind drug (blue triangle) in the lamellar bilayer. (b) M2(21–61) causes strong curvature to the VM membrane and binds the resulting small unilamellar vesicles, in which it adopts a conformation incompetent to bind drug. (c) M2(21–61) causes moderate curvature to DMPC bilayers, and the TM helix conformation is partially able to bind drug.