A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.

Abstract

We have examined the differential binding of Hck and Fyn to HIV‐1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full‐length Nef bound to Hck SH3 with the highest affinity reported for an SH3‐mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the ‘RT loop’, positioned close to conserved SH3 residues implicated in the binding of proline‐rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300‐fold weaker affinity for Hck SH3 than full‐length Nef.

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G

“…For a`consolidated ligand', a peptide which binds simultaneously to the SH2 and SH3 domains of the Abl kinase protein, an a nity of approximately 250 nM was measured . The a nity of the HIV Nef protein to the HckSH3 domain, determined by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), is also similar (Lee et al, 1995) but the corresponding Nef-peptide bound with a K d of 91 mM (measured by Trp-¯uorescence).…”

“…C3G, a guanine-nucleotide exchange factor for the GTPases Rap 1 A and B (Gotoh et al, 1995), contains four potential CrkSH3 binding motifs (CB1 to CB4) clustered in the central region of the protein (Knudsen et al, 1994;Tanaka et al, 1994). A 10-amino acid peptide corresponding to the CB1-motif was found to bind the CrkSH3(1) domain with a K d of 1.9 mM , a remarkably high a nity when compared to other SH3-interacting peptides of this length measured with tryptophan¯uorescence (Lee et al, 1995). A conserved lysine residue present in all C3G-CB motifs (consensus P-P-x-L-P-x-K) contributes greatly to the peptide and protein binding speci®city of the CrkSH3(1) domain.…”

HIV-1 Nef Triggers Macrophage Fusion in a p61Hck- and Protease-Dependent Manner

“…In macrophages, three kinases of the Src family are expressed: Hck, Lck/Yes-related novel protein, and Gardner-Rasheed feline sarcoma (51). Interaction of Nef with Hck has been described as the highest affinity among the Src family kinases (21). Nef triggers Hck activation, whereas it has no effect on Lck/Yes-related novel protein activity and Gardner-Rasheed feline sarcoma is not a target of Nef (52).…”

“…HIV-1 Nef interacts with the Src homology 3 (SH3) domains of Src PTKs through a proline-rich (P72xxP75) motif in its core domain, and substitution of a single amino acid in the Nef PxxP motif was sufficient to protect HIV-1 transgenic mice from the development of AIDS-like disease (19,20). Binding of Nef to SH3 domains can inhibit the T cell-specific Lck kinase activity or activates the phagocyte-specific kinase hematopoietic cell kinase (Hck) (21). The binding affinity between Nef and Hck is the highest among the SH3-mediated interactions (22,23); however, very little is known about the functional consequences of Hck activation by Nef in macrophages.…”

From negative factor to a critical role in virus pathogenesis: the changing fortunes of Nef

“…Using filterbinding assays and plasmon resonance it was shown that the Nef proline motif bound to the SH3 domain of Hck with high affinity (K D 250 nM for intact Nef but 300-fold weaker for the , p97, p75, p57, p55, p32, p28, p26 p70, p60, p56, p44, p36, p32, p30, p28, p27 Sawai et al (1994) Harris & Neil (1994) Rossi et al (1996) Harris ( Benichou et al (1994) Harris (1995) isolated PxxP motif) Lee et al, 1995). An additional PxxP motif at amino acids 147-150 was also implicated in the interaction, possibly explaining the decreased affinity of the isolated PxxP motif.…”