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 Trp21-Tyr22-Gly23. Our heteronuclear triple resonance NMR study of full length HAfp of sero subtype H1, solubilized in dodecylphosphatidyl choline (DPC), reveals a remarkably tight helical hairpin structure, with its N-terminal alpha-helix (Gly1-Glu11) packed tightly against its second alpha-helix (Trp14-Gly23), with six of the seven conserved Gly residues at the interhelical interface. The seventh conserved Gly residue in position 13 adopts a positive phi angle, enabling the hairpin turn that links the two helices. The structure is stabilized by multiple interhelical CaH to C=O hydrogen bonds, characterized by strong interhelical HN-Hand H-HNOE contacts. 15N 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.To enhance the resolution, we have recorded extensive residual dipolar coupling measurements in two different alignment media (polyacrylamide gel and liquid crystalline DNA) for both N-H and Ca-Ha interactions. Structures derived from the new data for the monomeric form of the fusion domain show many similarities but also distinct differences relative to those of earlier studies.
