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Presence of a helix in human CD4 cytoplasmic domain promotes binding to HIV-1 Nef protein.

Institut für Molekulare Biotechnologie, Beutenbergstrasse 11, D-07745 Jena, Germany.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 05/2002; 292(3):734-40. DOI: 10.1006/bbrc.2002.6700
Source: PubMed

ABSTRACT The Nef proteins of simian and human immunodeficiency viruses are known to directly bind and downregulate the CD4 receptor of infected cells. Recent results suggest that residues forming an alpha-helix N-cap in the CD4 cytoplasmic domain play a role in binding of CD4 to human immunodeficiency virus type 1 Nef protein. We determined the dissociation constants between Nef and several CD4 peptides that contain or do not contain the respective alpha-helix N-cap. Further, we compared helical secondary structure content of these CD4 peptide variants by circular dichroism spectroscopy. We conclude that presence of an alpha-helix in CD4 cytoplasmic domain increases CD4 affinity to Nef. In addition, the amino acid sequence of residues forming the helix N-cap influences CD4 affinity to Nef, too. Finally, the structural changes induced in Nef and CD4 upon binding to each other are investigated.

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    • "This could occur either by the association between Nef and clathrin-coated pits to juxtapose CD4 and the endocytotic machinery, or by the induction of a conformational change in the cytoplasmic tail to expose the di-leucine motif. A number of previous observations lend support to this hypothesis: firstly the binding of Nef to components of clathrin-coated pits (Greenberg et al., 1997; Lu et al., 1998); secondly the changes in secondary structure of CD4 peptides upon Nef binding as measured by CD spectra (Preusser et al., 2002); and lastly the fact that Nef-mediated CD4 down-modulation is independent of serine phosphorylation in the cytoplasmic tail (Garcia & Miller, 1991). Alternatively, the presence of the CD4 di-leucine motif might induce a conformational change in Nef, thus facilitating binding of Nef to endocytotic proteins. "
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    ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 205 residue, myristoylated phosphoprotein that has been shown to play a critical role in the replication and pathogenesis of the virus. One of the most studied functions of the Nef protein is the down-modulation of cell surface CD4. Nef has been reported to interact with both the cytoplasmic tail of CD4 and proteins that are components of the endocytic machinery, thereby enhancing the endocytosis of CD4 through clathrin-coated pits. A di-leucine motif in the cytoplasmic tail of CD4 (residues 413/414) was reported to be essential both for Nef mediated down-modulation and for Nef binding. In order to further characterize the involvement of this di-leucine motif in CD4 down-modulation we generated a CD4 mutant in which the leucines were substituted by alanines, termed CD4(LL-AA). We demonstrate here that, contrary to previous data obtained with the cytoplasmic tail of CD4 alone, full-length CD4(LL-AA) bound to Nef both in vivo, in recombinant baculovirus-infected Sf9 cells, and in vitro. In contrast the di-leucine motif was required for both Nef-mediated and phorbol ester-induced CD4 down-modulation, suggesting that the essential requirement for the di-leucine motif in CD4 down-modulation reflects the fact that this motif is needed for the interactions of CD4 with the endocytic machinery, not for the interaction with Nef. We have also exploited the observation that CD4(LL-AA) is refractory to Nef-mediated down-modulation to provide the first experimental evidence for a physical interaction between Nef and CD4 in intact mammalian cells.
    Journal of General Virology 11/2003; 84(Pt 10):2705-13. · 3.53 Impact Factor
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  • 12/2004: pages 269-286;