ausblenden:
Schlagwörter:
protein–protein interaction; structure prediction; directed evolution; yeast two-hybrid; random mutagenesis
Zusammenfassung:
Protein–protein interactions are critical to most biological processes, and locating protein–protein interfaces on protein structures is an important task in molecular biology. We developed a new experimental strategy called the ‘absence of interference’ approach to determine surface residues involved in protein–protein interaction of established yeast two-hybrid pairs of interacting proteins. One of the proteins is subjected to high-level randomization by error-prone PCR. The resulting library is selected by yeast two-hybrid system for interacting clones that are isolated and sequenced. The interaction region can be identified by an absence or depletion of mutations. For data analysis and presentation, we developed a Web interface that analyzes the mutational spectrum and displays the mutational frequency on the surface of the structure (or a structural model) of the randomized protein†. Additionally, this interface might be of use for the display of mutational distributions determined by other types of random mutagenesis experiments. We applied the approach to map the interface of the catalytic domain of the DNA methyltransferase Dnmt3a with its regulatory factor Dnmt3L. Dnmt3a was randomized with high mutational load. A total of 76 interacting clones were isolated and sequenced, and 648 mutations were identified. The mutational pattern allowed to identify a unique interaction region on the surface of Dnmt3a, which comprises about 500–600 Å2. The results were confirmed by site-directed mutagenesis and structural analysis. The absence-of-interference approach will allow high-throughput mapping of protein interaction sites suitable for functional studies and protein docking.
