The Conformation of Membrane-Associated HIV Nef and its Interactions with Hck

Nef is one of several HIV-1 accessory proteins and directly contributes to AIDS progression. Nef has no catalytic activity but instead realizes its functions by interacting with cellular proteins. Nef is myristoylated on the N-terminus, associates with membranes, and undergoes a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that conformational rearrangement enables membrane-associated Nef to interact with cellular proteins. Despite its obvious disease importance, there is little or no direct information about the conformation of membrane-bound Nef. In this work we used neutron reflection to reveal details of the conformation of membrane-bound Nef. The conformation of myristoylated Nef was studied upon binding to Langmuir monolayers of negatively-charged lipids. By adjusting the surface pressure, the extent of insertion of the myristate group could be controlled. At sufficiently high surface pressure such that the myristate group did not insert, adsorbed Nef was in a condensed state with the core domain directly against the lipid headgroups. At lower surface pressure such that the myristate group inserted into the membrane, adsorbed Nef was found to be in an extended state with the core domain displaced ∼ 70 A from the lipid headgroups. Thus, insertion of the myristate group triggers a conformational transition to an open configuration. This may have important ramifications for the ability of Nef to interact with host proteins. Prior work has shown that Nef interaction with the SH3 domains of Src-family kinases is a common early event in HIV-infected cells that generates important downstream signals essential for viral pathogenesis. We will report on the ability of membrane-bound Nef in each conformation to interact with the SH3 domain of Hck.