A basic patch on alpha-adaptin is required for binding of human immunodeficiency virus type 1 Nef and cooperative assembly of a CD4-Nef-AP-2 complex.
A critical function of the human immunodeficiency virus type 1 Nef protein is the downregulation of CD4 from the surfaces of infected cells. Nef is believed to act by linking the cytosolic tail of CD4 to the endocytic machinery, thereby increasing the rate of CD4 internalization. In support of this model, weak binary interactions between CD4, Nef, and the endocytic adaptor complex, AP-2, have been reported. In particular, dileucine and diacidic motifs in the C-terminal flexible loop of Nef have been shown to mediate binding to a combination of the alpha and sigma2 subunits of AP-2. Here, we report the identification of a potential binding site for the Nef diacidic motif on alpha-adaptin. This site comprises two basic residues, lysine-297 and arginine-340, on the alpha-adaptin trunk domain. The mutation of these residues specifically inhibits the ability of Nef to bind AP-2 and downregulate CD4. We also present evidence that the diacidic motif on Nef and the basic patch on alpha-adaptin are both required for the cooperative assembly of a CD4-Nef-AP-2 complex. This cooperativity explains how Nef is able to efficiently downregulate CD4 despite weak binary interactions between components of the tripartite complex.
Chaudhuri
Rittik
R
Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Building 18T, Room 101, National Institutes of Health, Bethesda, MD 20892, USA.
Mattera
Rafael
R
Lindwasser
O Wolf
OW
Robinson
Margaret S
MS
Bonifacino
Juan S
JS
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D015704:CD4 Antigens / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D015704:CD4 Antigens; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1