       Document 0661
 DOCN  M9620661
 TI    A single amino acid in the SH3 domain of Hck determines its high
       affinity and specificity in binding to HIV-1 Nef protein.
 DT    9602
 AU    Lee CH; Leung B; Lemmon MA; Zheng J; Cowburn D; Kuriyan J; Saksela K;
       Laboratory of Molecular Biophysics, Rockefeller University, New; York,
       NY 10021, USA.
 SO    EMBO J. 1995 Oct 16;14(20):5006-15. Unique Identifier : AIDSLINE
       MED/96067129
 AB    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.
 DE    *src Homology Domains  Amino Acid Sequence  Biosensors  Calorimetry
       Circular Dichroism  Comparative Study  Gene Products,
       nef/GENETICS/*METABOLISM  HIV-1/GENETICS/*METABOLISM  Models, Molecular
       Molecular Sequence Data  Oligopeptides/CHEMICAL SYNTHESIS  Precipitation
       Protein Binding  Protein Conformation  Protein-Tyrosine
       Kinase/GENETICS/*METABOLISM  Proto-Oncogene
       Proteins/GENETICS/*METABOLISM  Recombinant Proteins/METABOLISM  Sequence
       Homology, Amino Acid  Spectrometry, Fluorescence  Structure-Activity
       Relationship  Support, Non-U.S. Gov't  Support, U.S. Gov't, P.H.S.  Time
       Factors  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

