Extended Data Fig. 4. The 30-loop.

a, Potential interactions in the 30-loop are similar in both the neutral-pH (state I) and the extended HA2 (state IV) conformations. There are, however, several changes in the side chains involved in this interaction, owing to the relocation of the short helix of HA2 in the neutral-pH structure. This helix relocation removes an HA2 salt bridge between Arg54 of the short helix and Glu97 of the long helix, as well as the interaction of Lys51 with His106. These rearrangements permit new potential interactions between Thr30 of HA1 with Gln105 of HA2 and His106 of the adjacent HA2 chain. A salt bridge also forms between Lys27 of HA1 and Glu97 of HA2. b, Density of the 30-loop and interacting regions of the long helices of HA2 in the extended subtracted structure. c, The location and architecture of the 30-loop is conserved in all HAs, including influenza C HEF³⁷. In HA, a cluster of conserved hydrophobic-loop residues at positions 26, 34 and 36 packs against the strictly conserved Ile108 in the long α-helix; the strictly conserved Asn104 forms hydrogen bonds with loop residue Lys27 and Lys315 of HA1; and HA2 residue 105, conserved as Gln or Glu, interacts with Thr30. Amino-acid substitutions in the loop (Thr30 to Ser), the short α-helix (Arg54 to Lys) and the long α-helix (Gln105 to Lys and His106 to Ala) that interact with loop residues 28, 29 and 30, respectively, destabilize the mutant HAs, as shown by their elevated fusion pH^17,20. These observations indicate the functional importance of the loop and suggest its involvement in membrane fusion. Formation of the 180° turn in the extended helix requires removal of the 30-loop from its interactions with Gln105 and His106. The observed unfolding of the loop and its acquisition of susceptibility to protease digestion in stage V are consistent with this requirement and with the suggested role of the loop in supporting the extended helix in states II, III and IV.