Membrane Structures of the Hemifusion-Inducing Fusion Peptide Mutant G1S and theFusion-Blocking Mutant G1V of Influenza Virus HemagglutininSuggest a Mechanism for Pore Opening in MembraneFusion

Structures of G1S, G1V, and wild-type fusion domains in DPC micelles determined at pH 5 by ¹H-NMR spectroscopy. (A) 20 conformers representing the structure of G1S are shown in blue. For comparison, the“ most typical” conformer of the wild-type fusion domain is superimposed in red on the family of G1S conformers. (B) Twenty conformers representing the structure of G1V are shown in blue. For comparison, the “most typical” conformer of the wild-type fusion domain is superimposed in red on the family of G1V conformers. (C) Ribbon representation of the closest-to-the-mean conformer of the G1S structure with side chains inserted. (D) Ribbon representation of the closest-to-the-mean conformer of the G1V structure with side chains inserted. (E) Ribbon representation of the closest-to-the-mean conformer of the wild-type structure with side chains inserted. (F) End-on views of the N-terminal helices of wild-type, G1S, and G1V fusion domains. The first, second, third, and fourth turns of the G1S helix are labeled. (G) GRASP (44)-generated electrostatic surface potential representations of the structures of wild-type, G1S, and G1V fusion domains at pH 5. Negative, positive, and neutral potentials are shown in red, blue, and white, respectively. Side, top, and bottom views are shown for each structure, and several residues are labeled for reference.

Sections of NOESY spectra comparing NOEs that contribute to the definition of the different structures observed for the wild-type (WT), G1S, and G1V fusion domains in DPC micelles at pH 5. (A to C) NOE between Hα of Ile 10 and He3 of Trp 14 is present in the wild-type and G1S but absent in the G1V structure. (D to E) NOE between N-terminal Hα and HN of Phe 3 is present in G1S but very weak in the wild-type structure. (F to G) NOE between HN of Gly 13 and HN of Trp 14 is present in G1V but absent in the wild-type structure. All spectra were obtained under the same conditions and are plotted at the same contour level in each row. The thicker black lines are one-dimensional sections through the spectra at the positions of the indicated peaks.

Depth of three critical spin-labeled residues (Phe 3, Glu 11, and Ile 18) of wild-type, G1S, and G1V fusion domains in POPC:POPG (4:1) bilayers at pH 5. The depths were determined by fitting EPR spectra at increasing microwave powers in the presence of O₂, N₂, and NiEDDA to power saturation curves and ratioing the half-saturation powers in the presence of the different spin-relaxation agents as described in Materials and Methods. Inset: Best-fit calculated distances from NMR structures (abscissa) to experimental EPR depth parameters (ordinate) for wild-type, G1S, G1V, and four spin-labeled lipids used for depth calibration. All data fit the theoretical fitting function Φ = Atanh[B(x− C] + D, where A, B, C, and D are constants as described by Frazier et al. (19).

Wild-type(WT), G1S, and G1V fusion domain structures docked to POPC bilayers using the experimental depth data of Fig. 4. C termini are on the left and N termini are on the right. The polar lipid head groups and glycerol backbones are shown in orange, and the aliphatic side chains are shown in green in the molecular dynamic-simulated lipid bilayers.

ATR-FTIR spectra of wild-type, G1S, and G1V peptides bound to supported bilayers of POPC:POPG (4:1) at increasing concentrations at pH 5. Spectra were recorded after successive injections, from bottom to top, of 10, 20, 40, 80, and 100 μg/ml peptide and subsequent flushing of the cell with deuterium oxide buffer. The band at 1,735 cm⁻¹ arises from the lipid ester carbonyl groups, and the complex band between 1,600 and 1,700 cm⁻¹ is the amide I′ band from the bound fusion domains.