Rational Design and Protein Engineering of {SH2 Domain-≫ Flexible Linker-≫ Self-Controlling Peptide} Fusion System...

Self-controlling peptides (SCPs) are described as a specific sub-class of our previously proposed self-binding peptides (SBPs), which work as a molecular switch to control the protein function conversion between two or more biological states. In this study, a variety of artificial protein systems containing SCP with molecular switch functionality were rationally designed and systematically engineered by fusing several (weak and strong) phosphopeptide binders to the C-terminal tail of human Src SH2 domain via a flexible linker (FL), termed {SH2-> FL-> SCP} fusion protein systems. It is revealed that the intramolecular interaction between SH2 and SCP in the systems shares a similar binding behavior with the intermolecular interaction between SH2 and free phosphopeptide in structural and energetic points of view. In addition, FL is responsible for the binding dynamics of SCP to SH2, which restricts the SCP in a local region nearby SH2 binding site, thus equivalently enhancing the micro-concentration of SCP around the site, effectively increasing the collision frequency of SCP with the site, and consequently improving the apparent affinity of SCP to SH2. We carefully selected phosphopeptide binders and systematically optimized the sequence length and amino acid composition of FL, consequently resulting in a satisfactory {SH2-> poly(G)₁₂-> SCP(SIPM2-K_-2)} fusion protein system that was demonstrated to possess molecular switch functionality, in which the SCP binding and unbinding events were reversibly triggered by externally controlling its Tyr0-phosphorylation and pTyr0-dephosphorylation, respectively.