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. 2022 Aug 16;119(33):e2208011119.
doi: 10.1073/pnas.2208011119. Epub 2022 Aug 8.

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH

Eva Garcia-Moro  1 , Jie Zhang  2 , Lesley J Calder  1 , Nick R Brown  2 , Steven J Gamblin  2 , John J Skehel  2 , Peter B Rosenthal  1
Affiliations

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH

Eva Garcia-Moro et al. Proc Natl Acad Sci U S A. .

Abstract

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.

Keywords: cryo-EM; hemagglutinin; influenza; membrane fusion; protein folding.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Structural changes at low pH in HA0 and HA. (A) Molecular surface representations for HA0 at neutral pH, HA0 at low pH, and HA extended intermediate (state IV, PDB ID: 6Y5K) shown along the trimer axis (Top row) and side view (Bottom row). HA1 is colored in blue, and HA2 is in red with the fusion peptide (HA2 1 to 23) in yellow. Helix A (HA1 residues 38 to 55) is indicated by the white oval. At low pH, the HA1 membrane-distal domains move outward and the central coiled coil of HA2 extends between them, resulting in a similar conformation to that of the cleaved HA extended intermediate. (B) Ribbon diagram of a monomer colored as in A.
Fig. 2.
Fig. 2.
Structural changes at low pH in HA0 and HA highlighting HA2. Ribbon diagram of a monomer showing the conformational rearrangements in HA2 associated with the low-pH–induced transition of HA0 and HA. HA1 is in white; HA2 is colored by structural element: fusion peptide (1 to 23) in yellow, 24 to 37 in orange, A helix (38 to 55) in red, interhelical loop (56 to 75) in green, B helix in blue (76 to 105) and in pink (106 to 125), and C-terminal domain (126 to 172) in purple.
Fig. 3.
Fig. 3.
Comparison of the cleavage loop region in HA0 at neutral and low pH. (A) Uncleaved HA0 at neutral pH and (B) uncleaved HA0 at low pH. Monomers are shown in gray with N-terminal residues of HA1 in a darker gray. The cleavage loop (HA1 315 to HA2 23) is red. Arg329, the site of cleavage, is highlighted as a sphere. Glycans at Asn22 and Asn38 are in orange. In neutral-pH HA0, the cleavage loop is flexible and protrudes from the protein surface into the solvent, while in low-pH HA0 it is constrained to pack around the HA1 N terminus, which is disulfide bonded to HA2 (Cys14 to Cys137).
Fig. 4.
Fig. 4.
The structure of the fusion peptide in low-pH HA0. (A) A ribbon diagram of the fusion peptide (HA2 1 to 23) showing the solvent-exposed face. Glycine residues are labeled in red. (B) Closeup view of the three-turn amphipathic α-helical fusion peptide with the molecular surface of the binding site highlighting the pockets (green) for the side chains of fusion peptide residues W14, M17, I18, and W21. Side chains of HA1-contact residues (blue) and HA2 helices (red) are shown.
Fig. 5.
Fig. 5.
Extension of the coiled coil by threading. Surface and ribbon representations of HA0 showing side view (Top), and 180° rotated side view (Bottom). One monomer is colored with HA1 in blue and HA2 in red with the fusion peptide (HA2 1 to 23) in yellow. (A) For HA0 at neutral pH, helix A (HA1 residues 38 to 55), indicated by the white oval, is linked to the N terminus of the central coiled coil (HA2 residue 75) by the interhelical loop, which traverses a narrow channel between the membrane-distal domain and the central α-helical coiled coil. (B) In HA0 at low pH, the fusion peptide (yellow) is linked to the N terminus of the partly extended coiled coil (HA2 residue 50) by HA2 residues 24 to 49, which again traverse through the narrow channel between the now dilated membrane-distal domains and the central α-helical coiled coil. See also Movie S1.
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