Nef-mediated internalization of T-cell receptor molecules from the surface of an infected cell is required for the pathogenicity of HIV and disease progression to AIDS. This function depends on the N-terminal myristoylation of Nef, a lipid modification that targets the protein to membranes. We have analyzed how specific membrane properties and sequence motifs within Nef determine this interaction. Using time-resolved techniques we find that the association with membranes is a biphasic process with a fast rate for an electrostatic-driven protein-liposome interaction and a slow rate for the formation of an amphipathic helix. The rate of myristate insertion into liposomes depends on membrane curvature, while changes in the lipid composition with respect to phosphoinositides, cholesterol or sphingomyelin did not significantly alter the interaction. Moreover, Nef binding to membranes requires negatively charged liposomes, and mutations of basic and hydrophobic residues strongly diminished the association and changed the binding kinetics differently.
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We thank S. Gentz (Max Planck Institute for Molecular Physiology) for peptide synthesis, G. Holtermann for expert technical support and O. Fackler for stimulating discussions. F. Thorwirth, P. Verveer and P. Bastiaens are kindly acknowledged for help and advice with fluorescence imaging.
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Gerlach, H., Laumann, V., Martens, S. et al. HIV-1 Nef membrane association depends on charge, curvature, composition and sequence. Nat Chem Biol 6, 46–53 (2010). https://doi.org/10.1038/nchembio.268
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