Abstract: The N-terminal matrix domain of retroviral Gag, MA, is the key structural motif that mediates Gag membrane binding which leads to the formation of a two-dimensional protein lattice within the protein/membrane shell of budding virions. Several distinct mechanisms are implicated in MA membrane binding: electrostatic interactions between a patch of basic amino acids on MA and anionic lipids, hydrophobic interactions of MA’s myristoylated N-terminus with the bilayer, and specific binding to phosphatidylinositolbisphosphate, PI(4,5)P2, in the plasma membrane.
Here we use solid-supported tethered bilayer lipid membranes (stBLMs) to study structural, functional and thermodynamic aspects of Gag association with lipid bilayers and quantify MA binding to membranes of various compositions as a function of protein concentration. At ionic strengths where both electrostatic and hydrophobic interactions contribute to membrane association, a comparative study of –myrMA and +myrMA shows that myristoylation increases MA membrane association significantly, but its contribution to the Free Energy of binding is much less than the insertion energy of a free myristoyl chain into a membrane.
Our results argue against the hypothesis that PIP2 binding activates myristoyl exposure at the plasma membrane and disagrees in quantitative terms with vesicle (LUV) flotation assays from which it was concluded that MA dimerizes for effective membrane binding. However, protein-protein interactions between full-length Gag increase protein membrane affinity by two orders of magnitude. Cholesterol increases MA membrane binding significantly, both by facilitating membrane insertion of the myristate and protein binding to PI(4,5)P2. Structural investigations with neutron reflection of MA bound to stBLMs show the shallow penetration of the cationic protein surface into the bilayer and determine the orientation of the protein on the membrane surface.
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