The mechanism for low-pH-induced clustering of phospholipid vesicles carrying the HA2 ectodomain of influenza hemagglutinin.

Homotrimeric hemagglutinin (HA) is one of the major spike membrane glycoproteins of the influenza virus. Initial pH-triggered conformational changes in the target membrane-interacting HA2 domain are necessary for a preliminary step in membrane fusion. Using spin-labeling electron paramagnetic resonance (EPR) spectroscopy, we examined subsequent pH-dependent changes of a membrane-bound HA2 construct (FHA2, aa 1-127). Residues 91-94, 108-115, 122, and 125 were mutated to cysteine and spin-labeled. Low solvent accessibility and side chain mobility were observed by EPR at positions 91-94, 122, and 125. Spin-labels at residues 108-115 were solvent-exposed and highly mobile, revealing the presence of a flexible loop. These results are consistent with the low-pH crystal structure of a truncated HA2 domain, particularly the unusual kink loop at residues 108-115 [Bullough et al. (1994) Nature (London) 371, 37-43]. Most interestingly, at endosomal pH, spin-labels at 108-115 become immobile and no longer solvent-exposed, and this change is reversible upon reneutralization. However, little change in the EPR line shape and accessibility of spin-labels was observed in other regions. This observation implies that the FHA2 trimers interact reversibly via this specific loop, most likely in an intermolecular fashion. Furthermore, this interaction correlates well with a reversible pH-dependent clustering of FHA2-bearing vesicles evidenced by the reversible increase in turbidity and further confirmed in detail by electron microscopy. The implications of this reversible, pH-dependent interaction between FHA2 trimers are discussed in light of recent fusion models.