The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding surface by NMR

Using heteronuclear NMR spectroscopy, we demonstrate that a 13-residue peptide (MSQIKRLLSEKKT) from the cytoplasmic tail of CD4 binds to Nef protein. This part of CD4 is critical for downregulation of CD4 by HIV-1 Nef [Aiken el al. (1994) Cell 76, 853-864]. We show that a control peptide without the central dileucine does not bind to Nef. The dependence of Nef H-1 and N-15 amide chemical shifts on peptide concentration indicates that the binding is in the fast chemical exchange limit, with a dissociation constant K-d of similar to 1 mM, The peptide binding site has been mapped onto the previously determined solution structure of HIV-1 Nef [Grzesiek ct al. (1996) Nat. Struct. Biol. 3, 340-345] on the basis of peptide-induced chemical shift changes. It comprises amino acids W57, L58, E59, G95, G96, L97, R106, and L110. When Nef is complexed to the SH3 domain of Hck tyrosine protein kinase, the peptide binds to the same site on Nef but with slightly higher affinity (K-d similar to 0.5 mM). This indicates that the binding of CD4 and Hck SH3 to Nef are two compatible and slightly cooperative events.