Predicting Functional Interactions in the Influenza Hemagglutinin Transmembrane Doman via Simulation

The precise mechanism of cell entry by influenza remains poorly understood, despite many years of research. Entry into the cytoplasm is preceded by a membrane fusion event between the virion and host endosomal membrane, mediated by the fusion protein hemagglutinin. Hemagglutinin resides in the viral membrane with one transmembrane helix. Its ectodomain is trimeric, and this enforces close proximity between the three helices in the viral membrane. Previous research has demonstrated that the transmembrane domain of hemagglutinin is crucial to its pathogenicity, and that while some mutations are allowed, productive viral fusion to the target membrane is blocked by drastic deletions or replacement with a GPI anchor. We have conducted molecular dynamics simulations of the hemagglutinin transmembrane domain to understand any interactions that may occur between the domains and postulate how they can impact hemagglutinin function.

Our simulations indicate that the helices can associate in a membrane without the ectodomain present. We have used a multi-scale simulation approach to examine the stability of encounter complexes in order to identify key interactions and predict changes that would disrupt them. First, coarse-grained simulations were used to generate a large population of encounter complexes between helices, which were then clustered to create a diverse sample set. The resulting complexes were simulated at atomic resolution to test their stability and identify specific interactions in the membrane.

At atomic resolution, simulations show that transmembrane domain complexes remain stable in the membrane environment and exhibit key hydrophobic interactions between residues in the membrane-inserted region of the peptide. In order to test their importance, we created two additional sets of all-atom simulations. The F205A mutant simulations were designed to ablate the most probable single contact between monomers, while the L198A V201A F205A W208A mutant simulations aimed to more broadly disrupt common inter-peptide interaction modes. Surprisingly, our simulations show that both sets of mutants exhibit similar stability in the membrane environment despite severely diminished interaction at the mutation sites. In contrast, simulations of a previously studied deletion mutant show a substantial disruption in trimer stability and membrane disruption. In order to further quantify trimeric stability in the membrane, a series of pulling simulations aims to determine the relative free energy of dissociation for the wild type and quadruple mutant trimeric complexes.