A novel hSH3 domain scaffold engineered to bind folded domains in CD2BP2 and HIV capsid protein.
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Engineered protein scaffolds destined to target and inhibit molecular interactions in the context of disease bear great diagnostic and therapeutic potential. Apart from antibodies several alternative scaffolds have been exploited over the years making use of the fact that individual domain families are best suited for certain target families. Here we capitalize on the helically extended SH3 domain hSH3N of the ADAP protein as loop-randomized template that was tested against HIV capsid (CA) protein and the GYF domain of human CD2BP2 as molecular targets. Phage display of mutant proteins resulted in the isolation of variants with changes in all randomized positions compared with wild-type hSH3N. The soluble scaffolds bind with 340 and 600 nM affinity to CA and CD2BP2, respectively, and employ large molecular surfaces to pull out these targets from complex mixtures. (+info)
Superbinder SH2 domains act as antagonists of cell signaling.
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Protein-ligand interactions mediated by modular domains, which often play important roles in regulating cellular functions, are generally of moderate affinities. We examined the Src homology 2 (SH2) domain, a modular domain that recognizes phosphorylated tyrosine (pTyr) residues, to investigate how the binding affinity of a modular domain for its ligand influences the structure and cellular function of the protein. We used the phage display method to perform directed evolution of the pTyr-binding residues in the SH2 domain of the tyrosine kinase Fyn and identified three amino acid substitutions that critically affected binding. We generated three SH2 domain triple-point mutants that were "superbinders" with much higher affinities for pTyr-containing peptides than the natural domain. Crystallographic analysis of one of these superbinders revealed that the superbinder SH2 domain recognized the pTyr moiety in a bipartite binding mode: A hydrophobic surface encompassed the phenyl ring, and a positively charged site engaged the phosphate. When expressed in mammalian cells, the superbinder SH2 domains blocked epidermal growth factor receptor signaling and inhibited anchorage-independent cell proliferation, suggesting that pTyr superbinders might be explored for therapeutic applications and useful as biological research tools. Although the SH2 domain fold can support much higher affinity for its ligand than is observed in nature, our results suggest that natural SH2 domains are not optimized for ligand binding but for specificity and flexibility, which are likely properties important for their function in signaling and regulatory processes. (+info)
Graphical analysis of flow cytometer data for characterizing controlled fluorescent protein display on lambda phage.
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