THE CRYSTAL-STRUCTURE OF HIV-1 NEF PROTEIN-BOUND TO THE FYN KINASE SH3 DOMAIN SUGGESTS A ROLE FOR THIS COMPLEX IN ALTERED T-CELL RECEPTOR SIGNALING

Background: Human immunodeficiency virus (HIV) Nef protein accelerates virulent progression of acquired immunodeficiency syndrome (AIDS) by its interaction with specific cellular proteins involved in signal tra nsduction and host cell activation, Nef has been shown to bind specifi cally to a subset of the Src family of kinases. The structures of free Nef and Nef bound to Src homology region 3 (SH3) domain are important for the elucidation of how the affinity and specificity for the Src k inase family SH3 domains are achieved, and also for the development of potential drugs and vaccines against AIDS. Results: We have determine d the crystal structures of the conserved core of HIV-I Nef protein al one and in complex with the wild-type SH3 domain of the p59(fyn) prote in tyrosine kinase (Fyn), at 3.0 Angstrom resolution. Comparison of th e bound and unbound Nef structures revealed that a proline-rich motif (Pro-x-x-Pro), which is implicated in SH3 binding, is partially disord ered in the absence of the binding partner; this motif only fully adop ts a left-handed polyproline type II helix conformation upon complex f ormation with the Fyn SH3 domain. In addition, the structures show how an arginine residue (Arg77) of Nef interacts with Asp 100 of the so-c alled RT loop within the Fyn SH3 domain, and triggers a hydrogen-bond rearrangement which allows the loop to adapt to complement the Nef sur face. The Arg96 residue of the Fyn SH3 domain is specifically accommod ated in the same hydrophobic pocket of Nef as the isoleucine residue o f a previously described Fyn SH3 (Arg96 --> IIe) mutant that binds to Nef with higher affinity than the wild type. Conclusions: The three-di mensional structures support evidence that the Nef-Fyn complex forms i n vivo and may have a crucial role in the T cell perturbating action o f Nef by altering T cell receptor signaling. The structures of bound a nd unbound Nef reveal that the multivalency of SH3 binding may be achi eved by a ligand induced flexibility in the RT loop. The structures su ggest possible targets for the design of inhibitors which specifically block Nef-SH3 interactions.