SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef
| Autor: | M, Hiipakka, K, Poikonen, K, Saksela |
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| Rok vydání: | 1999 |
| Předmět: |
Models
Molecular Protein Conformation Protein-Tyrosine Kinases Arginine Ligands Protein Engineering Binding Competitive Gene Products nef Substrate Specificity src Homology Domains Amino Acid Substitution Peptide Library Proto-Oncogene Proteins Mutation HIV-1 Proto-Oncogene Proteins c-hck Thermodynamics Amino Acid Sequence nef Gene Products Human Immunodeficiency Virus Cloning Molecular |
| Zdroj: | Journal of molecular biology. 293(5) |
| ISSN: | 0022-2836 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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