Helical hairpin structure of influenza hemagglutinin fusion peptide stabilized by charge-dipole interactions between the N-terminal amino group and the second helix

Abstract

The fusion domain of the influenza coat protein hemagglutinin HA2, bound to dodecyl phosphocholine micelles, was recently shown to adopt a structure consisting of two antiparallel α-helices, packed in an exceptionally tight hairpin configuration. Four interhelical H(α) to C═O aliphatic H-bonds were identified as factors stabilizing this fold. Here, we report evidence for an additional stabilizing force: a strong charge-dipole interaction between the N-terminal Gly(1) amino group and the dipole moment of helix 2. pH titration of the amino-terminal (15)N resonance, using a methylene-TROSY-based 3D NMR experiment, and observation of Gly(1 13)C' show a strongly elevated pK = 8.8, considerably higher than expected for an N-terminal amino group in a lipophilic environment. Chemical shifts of three C-terminal carbonyl carbons of helix 2 titrate with the protonation state of Gly(1)-N, indicative of a close proximity between the N-terminal amino group and the axis of helix 2, providing an optimal charge-dipole stabilization of the antiparallel hairpin fold. pK values of the side-chain carboxylate groups of Glu(11) and Asp(19) are higher by about 1 and 0.5 unit, respectively, than commonly seen for solvent-exposed side chains in water-soluble proteins, indicative of dielectric constants of ε = ∼30 (Glu(11)) and ∼60 (Asp(19)), placing these groups in the headgroup region of the phospholipid micelle.

Figure 1

pH titration of the N-terminal amino ¹⁵N resonance of Gly¹ with the HACAN CH₂-TROSY experiment. (A) Superimposed small regions of ¹H/¹⁵N cross sections taken through the 3D CH₂-TROSY HACAN spectrum of HAfp23, showing the correlation between the Gly^{1 1}H^α2/¹H^α3 and amino ¹⁵N chemical shifts. (B) The Gly¹ backbone amino ¹⁵N (black) and ¹³C^α (red) chemical shift dependence on pH is shown. A non-linear least-squares regression was used to fit the Henderson-Hasselbalch equation to the titration curves. The fitted values are: pK = 8.73±0.07, ¹⁵N and ¹³C^α chemical shifts of 27.0±0.6. ppm and 44.3±0.1 ppm, respectively, for the protonated form (NH₃⁺), and 13.6±0.4 ppm and 46.8±0.1 ppm, respectively, for the deprotonated form (NH₂). The fitted pK value when measuring the Gly^{1 13}C′ shift on a separate sample (Fig. 2) is 8.84±0.05.

Figure 2

Changes in the ¹³C′ chemical shifts on titrating the amino group of Gly¹. (A) Superimposed small regions of cross-sections taken through the 3D HCACO spectra of HAfp23, recorded at pH values ranging from pH 7.09 to 9.62, with ¹³C′ chemical shift changes summarized in (C). The Gly^{8 13}C′ chemical shifts could not be identified uniquely due to spectral congestion and are not reported. (B) pH dependence of Gly^{1 13}C′ (green, right axis) and ¹³C′ chemical shifts of various residues near the end of helix 2. Reported chemical shift changes are relative to values measured at pH 7.09. Traces shown represent nonlinear regression fits to the Henderson-Hasselbalch equation, yielding: Δδ = 7.1±0.2 ppm and pK = 8.84±0.05 for Gly¹; Δδ = −0.08±0.02 ppm and pK = 8.9±0.2 for Ile¹⁸; Δδ = −0.46±0.02 ppm and pK = 8.84±0.09 for Gly²⁰; Δδ = −0.35±0.06 ppm and pK = 8.9±0.2 for Trp²¹; Δδ = +0.37±0.04 ppm and pK = 8.87 ±0.10 for Gly²³.

Figure 3

Chemical shift titrations of the carboxylate groups of Glu¹¹ and Asp¹⁹. (A) Superimposed small regions of cross sections taken through the 3D HCCO spectra taken over the range from pH 6.5 to 2.9, showing the correlation between the carboxylic acid ¹³C and its vicinal methylene ¹H chemical shifts. Chemical shift versus pH for (B) the Glu¹¹ side chain ¹³C^δ (black) and ¹³C^γ (red), and (C) the Asp¹⁹ side chain ¹³C^γ (black) and ¹³C^β (red) are fit to the Henderson-Hasselbalch equation using non-linear least-squares regression. Fitted values for (B) are: pK = 5.31±0.01, with ¹³C^δ and ¹³C^γ chemical shift of 179.2 ppm and 33.0 ppm, respectively, for the protonated form (COOH), and a ¹³C^δ and ¹³C^γ chemical shift of 183.2 ppm and 36.3 ppm for the deprotonated form (COO⁻). Fitted values for Asp¹⁹ are: pK = 4.35±0.02, with ¹³C^γ and ¹³C^β chemical shift of 175.5 ppm and 37.1 ppm, respectively, for the protonated form, and ¹³C^γ and ¹³C^β chemical shifts of 178.8 ppm and 40.3 ppm for the deprotonated form (COO⁻).