title: SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef.
abstract: The avid binding of HIV-1 Nef to the Src homology-3 (SH3) domain of Hck (KD 250 nM) has been shown to involve an interaction between the RT-loop of Hck-SH3 and residues in Nef outside of its prototypic polyproline type II (PPII) helix-containing SH3-ligand region. Such distinctive interactions are thought to provide specificity and affinity for other SH3/ligand protein complexes as well. Here, we have constructed and successfully displayed on the surface of M13 bacteriophage particles a complex library of SH3 domains, which are derived from Hck but carry a random hexapeptide substitution in their RT-loops (termed RRT-SH3). Using this strategy we have identified individual RRT-SH3 domains that can bind to Nef up to 40-fold more avidly than Hck-SH3. Some of these high-affinity RRT-SH3 domains resembled Hck-SH3 in that they bound much less well to a Nef variant containing an engineered F90R mutation that interferes with docking of the native Hck RT-loop. In addition, we could also select RRT-SH3 domains with an opposite specificity, which were dependent on the Arg90 residue for strong binding, and bound 100-fold less well to unmodified Nef. These results demonstrate the utility of phage-display in engineering of signaling protein interaction domains, and emphasize the importance of the RT-loop in SH3 ligand selection, thus suggesting a general strategy for creating SH3 domains with desired binding properties.
authors.affiliation: Institute of Medical Technology, University of Tampere, Tampere, FIN-33101, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Hiipakka
authors.forename: K
authors.initials: K
authors.lastname: Poikonen
authors.forename: K
authors.initials: K
authors.lastname: Saksela
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D003001:Cloning, Molecular; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051572:Proto-Oncogene Proteins c-hck; D013379:Substrate Specificity; D013816:Thermodynamics; D054311:nef Gene Products, Human Immunodeficiency Virus; D018909:src Homology Domains
chemical_list: D015693:Gene Products, nef; D008024:Ligands; D019151:Peptide Library; D011518:Proto-Oncogene Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D001120:Arginine; D011505:Protein-Tyrosine Kinases; D051572:Proto-Oncogene Proteins c-hck