Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity

Qiao, Hui; Pelletier, Sandra L.; Hoffman, Lucas; Hacker, Jill; Armstrong, R. Todd; White, Judith M.

We have conducted a preliminary analysis to assess whether the fusion defects observed with V55P and V55P/ S71P have structural correlates (Armstrong, R.T., and J.M. White, unpublished results). To do this, we engineered the Pro mutations into their corresponding locations in an Escherichia coli expression vector that encodes HA2 residues 33 to 127 (gift of Dr. Peter Kim, Whitehead Institute, Cambridge, MA). The mutant proteins were expressed, purified to homogeneity, and analyzed by circular dichroism. Given their fusion phenotypes as full-length HA proteins (see above) and the predicted general destabilizing effect of prolines on helix formation (O'Neil and Degrado, 1990; Lupas et al., 1991; Lupas, 1996) , we predicted that the order of helix-forming potential would be: wt Ͼ S71P Ͼ L80P Ͼ V55P Ͼ V55P/S71P. The predicted order based solely on the COILS 2 scores is: wt Ͼ S71P Ͼ V55P Ͼ L80P Ͼ V55P/S71P. The only difference between the two predictions is the relative rankings of V55P and L80P. The helix-forming potentials of the E. coli constructs at pH 5 were: wt ϭ 77%, S71P ϭ 55%, V55P ϭ 38%, L80P ϭ 35%, and V55P/S71P ϭ 28%. Hence the observed order for helix forming potential (also seen at pH 7) is in good agreement with the structural predictions.