The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface

Abstract

All but five of the N-terminal 23 residues of the HA2 domain of the influenza virus glycoprotein hemagglutinin (HA) are strictly conserved across all 16 serotypes of HA genes. The structure and function of this HA2 fusion peptide (HAfp) continues to be the focus of extensive biophysical, computational, and functional analysis, but most of these analyses are of peptides that do not include the strictly conserved residues Trp 21 -Tyr 22 -Gly 23 . The heteronuclear triple resonance NMR study reported here of full length HAfp of sero subtype H1, solubilized in dodecylphosphatidyl choline, reveals a remarkably tight helical hairpin structure, with its N-terminal α-helix (Gly 1 -Gly 12 ) packed tightly against its second α-helix (Trp 14 -Gly 23 ), with six of the seven conserved Gly residues at the interhelical interface. The seventh conserved Gly residue in position 13 adopts a positive ϕ angle, enabling the hairpin turn that links the two helices. The structure is stabilized by multiple interhelical C α H to C¼O hydrogen bonds, characterized by strong interhelical H N -H α and H α -H α NOE contacts. Many of the previously identified mutations that make HA2 nonfusogenic are also incompatible with the tight antiparallel hairpin arrangement of the HAfp helices. 15 N relaxation analysis indicates the structure to be highly ordered on the nanosecond time scale, and NOE analysis indicates HAfp is located at the water-lipid interface, with its hydrophobic surface facing the lipid environment, and the Gly-rich side of the helix-helix interface exposed to solvent. Of the three different types of RNA influenza virus, the influenza A type is the most common and virulent human pathogen. Based on the antibody response to its viral surface proteins, hemagglutinin and neuraminidase, influenza type A is further subdivided into serotypes, ranging from subtypes H1-16 for hemagglutinin, and N1-9 for neuraminidase. Subtypes H1, H2, H3, N1, and N2 are the most common in human disease (1). Whereas neuraminidase is an enzyme responsible for cleavage of cellular sialic acid carbohydrates, a key step in viral exit, HA mediates cellular entry through binding to carbohydrates on epithelial cells and subsequent delivery of the viral contents to the cellular interior (2, 3).Prior to infection of human tissue, the homotrimeric hemagglutinin, HA0, requires cleavage by one of several human proteases (4), resulting in a trimer of heterodimeric proteins, HA1 and HA2, covalently linked to one another by a single disulfide bridge. The atomic structures of the precursor and processed HA have been solved by X-ray crystallography (5-7) and reveal that the main rearrangement taking place upon cleavage is the relocation of the newly formed N-terminal HA2 tail from the surface of HA0 to the helical core of the HA2 ecto domain. Upon lowering the pH, the N-terminal HA2 fusion domain is extruded from the HA2 helical core to a position where it can target the endosomal membrane, while the C-terminal transmembrane helix keeps HA2 anchored to the viral membrane. ...

Plasma Membrane Localization of Solanum tuberosum Remorin from Group 1, Homolog 3 Is Mediated by Conformational Changes in a Novel C-Terminal Anchor and Required for the Restriction of Potato Virus X Movement

“…The predicted structure has a high helical content, therefore probably corresponding to the conformation adopted by RemCA in hydrophobic environments. This hypothesis is consistent with the structure of the HAfp, which forms a tight hairpin of a-helices in the presence of lipids (Lorieau et al, 2010). The distribution of the relative hydrophobicity of amino acids reveal an accumulation of hydrophobic amino acids at the center of the hairpin, with the side chains of hydrophiliccharged amino acids pointing outward (Fig.…”

“…2C; Supplemental File S1). This structure is reminiscent of a recently published structure for influenza hemagglutinin fusion peptide (HAfp; Lorieau et al, 2010;Supplemental Fig. S2).…”

Transmembrane orientation and possible role of the fusogenic peptide from parainfluenza virus 5 (PIV5) in promoting fusion

“…However, the hydrophobic region of the FP in the intact virus spans residues up to Arg25, and NMR studies have been conducted with peptides spanning between 20 to 23 residues, with an artificial oligo-Lys tail added to enhance water solubility. The dynamics and conformational properties of the 20-versus 23-residue peptide differ significantly (18), as expected for a finely tuned system with multiple low-lying energy wells that are progressively populated during fusion. These distinct structural states, and their sensitivity to small changes in sequence and environment, may be both functionally relevant and reflect the energetic fine-tuning of the landscape and the dynamic nature of fusion.…”

“…For example, the N-terminal peptide of gp41 has been reported to adopt a TM (15), tilted (28)(29)(30), and beta (19,(26)(27)(28)31) conformation in various membrane mimetics. Synthetic versions of the FP from influenza virus HA2 span approximately half of the bilayer width, but as a bent helix (12) or helical hairpin (18) in micelles. However, the hydrophobic region of the FP in the intact virus spans residues up to Arg25, and NMR studies have been conducted with peptides spanning between 20 to 23 residues, with an artificial oligo-Lys tail added to enhance water solubility.…”

A bundling of viral fusion mechanisms

“…NMR structures of influenza fusion peptides in micelles show a kinked helix (9) or a helical hairpin (10). Whether these conformations change upon formation of a peptide assembly is, of course, an open question.…”