A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.
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.
Lee
C H
CH
Laboratory of Molecular Biophysics, Rockefeller University, New York, NY 10021, USA.
Leung
B
B
Lemmon
M A
MA
Zheng
J
J
Cowburn
D
D
Kuriyan
J
J
Saksela
K
K
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D002151:Calorimetry; D011232:Chemical Precipitation; D002942:Circular Dichroism; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D051572:Proto-Oncogene Proteins c-hck; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013997:Time Factors; D054311:nef Gene Products, Human Immunodeficiency Virus; D018909:src Homology Domains
D015693:Gene Products, nef; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D011505:Protein-Tyrosine Kinases; D051076:Proto-Oncogene Proteins c-fyn; D051572:Proto-Oncogene Proteins c-hck