Computational analysis and protein engineering of artificial PDZ domain/ self-binding peptide fusion biomacromolecular system with molecular switch functionality

Self-binding peptides (SBPs) are peptide-dependent intramolecular interactions described by our group, which conceptualize the short peptide segment within a monomeric protein as able to perform biological function by its dynamic and reversible binding to a cognate domain partner in the same monomer. Previously, we offered evidences that the SBPs represent a new biomolecular dynamic phenomenon that works across folding and binding, and also proposed the SBPs as potential drug targets for pharmacological intervention. Here, we attempted to model and design artificial protein systems containing SBP with molecular switch functionality by using computational peptidology strategies and protein engineering methods. In the procedure, the free decapeptide ligands were fused to the C-terminal tail of human CAL PDZ domain through a flexible polypeptide linker, thus resulting in a number of PDZ/SBP fusion biomacromolecular systems. The intramolecular binding event of SBP to PDZ was observed in the fusion systems as well as between the free decapeptide and PDZ. We carefully examined linker effects on the intramolecular binding event and systematically optimized the length and composition of linker to improve binding. Computational analysis suggested dynamics, but not thermodynamics, primarily responsible for the binding of SBP to PDZ. The linker plays an important role, as it restrains the SBP within a small local spatial region nearby the binding site of PDZ. The term effective concentration (EC) was defined to characterize the high local concentration of SBP at a small region due to the linker restriction, which improves the binding probability of SBP to PDZ from a dynamics point of view. The CAL PDZ/SBP(iCAL36-K-6) fusion protein system with poly(G)8 linker can work as designed well, where the SBP(iCAL36-K-6) dynamically binds to/unbinds from PDZ in a regulatable manner by externally controlling its C-terminal deamidation/amidation, thus exhibiting a typical molecular switch functionality.