The Conformation and Activity of Membane-Bound HIV Nef Studied by Neutron Reflectivity

Abstract

Nef is one of six 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 may undergo 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 membrane-bound Nef, and its ability to bind to the SH3 domain of Hck, was found to depend upon the mode of membrane binding. In solution Nef in known to bind the SH3 domain of Hck with high affinity (kd = 250 nM). In one mode, Nef was bound through an N-terminal His tag to Langmuir monolayers of DPPC mixed with a synthetic metal-chelating lipid (Biophys. J. 99, 1940, 2010). In that case, for a range of pH and salt concentration the core domain of membrane-bound Nef lies within a few angstroms of the lipid headgroups and a portion of the protein, presumably from the N-terminal arm, is inserted. In this conformation, Nef does not bind the SH3 domain of Hck. However, upon binding to negatively-charged membranes in absence of an interaction through the N-terminus, the core domain of Nef is displaced from the membrane, and binding of SH3 occurs. The ramifications of the these results for the actions of Nef in-vivo will be discussed.