Stability of HA2 Prefusion Structure and pH-Induced Conformational Changes in the HA2 Domain of H3N2 Hemagglutinin

Abstract

Influenza hemagglutinin is the fusion protein that mediates fusion of the viral and host membranes through a large conformational change upon acidification in the developing endosome. The "spring-loaded" model has long been used to describe the mechanism of hemagglutinin and other type 1 viral glycoproteins. This model postulates a metastable conformation of the HA2 subunit, caged from adopting a lower-free energy conformation by the HA1 subunit. Here, using a combination of biochemical and spectroscopic methods, we study a truncated construct of HA2 (HA2*, lacking the transmembrane domain) recombinantly expressed in Escherichia coli as a model for HA2 without the influence of HA1. Our data show that HA2* folds into a conformation like that of HA2 in full length HA and forms trimers. Upon acidification, HA2* undergoes a conformational change that is consistent with the change from pre-to postfusion HA2 in HA. This conformational change is fast and occurs on a time scale that is not consistent with aggregation. These results suggest that the prefusion conformation of HA2 is stable and the change to the postfusion conformation is due to protonation of HA2 itself and not merely uncaging by HA1.

Influenza A virus hemagglutinin: from classical fusion inhibitors to proteolysis targeting chimera-based strategies in antiviral drug discovery

“…Furthermore, these studies have examined how structural properties, such as stalk height and secondary-site binding, affect the interaction with SA, and the interplay between SA binding sites in HA and NA in the attachment to the host cell receptors. Similarly, beyond the static spring-loaded fusion model of HA-mediated membrane fusion, both structural, spectroscopic, and computational studies are providing insights into the large-scale conformational rearrangements implicated in the release of the FP and remodeling of HA 2 , and the molecular mechanisms that underlie the acidification-promoted fusion of viral and cell membranes [57][58][59][60][61][62].…”

Molecular dynamics investigation of the influenza hemagglutinin conformational changes in acidic pH

“…25 Alternatively, upon acidification, the major conformational change happen in HA2. The B-loop located between two helices in HA2 has a loop-to-helix transformation and forms an α-helix elongating the fusion peptide to the endosomal membrane [26][27][28] (Fig. 1).…”