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.

Substitutions were analyzed by calculating the predicted relative stability of the resulting coiled-coils using the COILS 2 algorithm employed by Kim and co-workers (Carr and Kim, 1993) ; the score for wt-HA2 residues 54-81 is 1.62. In addition, the predicted ability of each site to accommodate the planned mutation in the neutral-pH form of BHA was considered as follows: The dihedral angles of the wild-type residue at each location in the native structure were determined. As indicated in Table I , substitution of Ala, Gly, and Pro gave acceptable phi and psi angles at all three positions. Next we made, computationally, isomorphous substitutions of the mutant residues in HA2 54-81 in the native structure (Watowich et al., 1994) and calculated the internal energy of the segment as described in Materials and Methods. As listed in Table I , all of the proposed substitutions except L80P were predicted not to change the internal energy of this region significantly. Calculations for L80P indicated a significant increase in internal energy due to a predicted close approach between hydrogen atoms of the main chain and the introduced Pro. The low-pH conformational change in HA is believed to represent a two-state system under kinetic control (Baker and Agard, 1994) . Therefore, a mutation that affects the height of the energy barrier between the two states would be predicted to change the kinetics of fusion. Since coiledcoil formation is involved in this transition (Carr and Kim, 1993; Bullough et al., 1994) and since prolines destabilize coiled-coils (O'Neil and Degrado, 1990; Lupas et al., 1991) , we predicted that Pro mutations would affect the rate of fusion. If the mutations also abrogated the formation of the final fusogenic state, then the overall extent of fusion would also be compromised. We therefore predicted that Pro mutations, especially those at "d" positions (V55P and L80P), would inhibit the rate or extent of fusion. We further predicted that an HA with two Pro mutations (V55P/S71P) would be severely compromised for fusion.