Fusion Protein TM-TM Interactions: Modulators of Pre-Fusion Protein Stability

Abstract

Membrane associated domains of proteins are often under-studied because of their lipid environment, however recent studies suggest that protein transmembrane domains (TMDs) are important for cell signaling events, and protein oligomerization. Enveloped viruses utilize fusion proteins (F) studding the envelope of the virus to promote fusion of the viral envelope with a target cell membrane. To drive fusion, the F proteins undergo large conformational changes, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion conformation; understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein TMDs have implicated the TMD in the fusion process, but the structural and molecular details of the role of TMDs in fusion remains unclear. We previously utilized analytical ultracentrifugation to demonstrate that isolated TMDs of Hendra virus F associate in a monomer-trimer equilibrium. To determine elements critical for this association, we examined the Hendra virus F sequence. Directly upstream of the TMDs is a heptad repeat domain that contains a leucine-isoleucine zipper (LIZ) which continues in frame into the TMD. We found that a TMD replacing four L/I residues with alanine dramatically reduced TM-TM association, implicating the LIZ in TM-TM interactions. Studies with the LIZ mutations in the whole protein indicate decreased protein stability, suggesting TM-TM interactions promoted by the LIZ may be critical for pre-fusion stability. Previous studies demonstrated that the pre-fusion conformation of F can be thermally triggered to its post-fusion conformation. We used this characteristic of F to demonstrate that reduced TM-TM association in the LIZ F mutant results in reduced stability of F in its pre-fusion conformation. Together our data indicate that the TMD is important in F protein stability and triggering.