Manipulation of protein-complex function by using an engineered heterotrimeric coiled-coil switch

Design methodology of variant proteins, in which original functions can be manipulated by additive ligand binding, is an attractive target of protein engineering. Especially for multi-protein complexes, techniques for constructing variants which allow the switching on or off of original functions by ligands have been limited until now. We examined a method of utilizing a de novo designed protein module, IZ-DS, which has a tertiary structure that can be significantly changed from a random coil to a folded coiled-coil structure following binding with peptide ligand, IZ-3K. By introducing a metamorphosis IZ-DS sequence to one of the components in a target multi-protein complex, the IZ-3K binding and the subsequent structural transition of the IZ-DS moiety would affect the tertiary structure of the introduced protein unit, and the function of the total multi-protein complex may also be altered. In this research, we used the T7 RNA polymerase (T7 RNAP)/T7 lysozyme complex as the target multi-protein complex, in which allosteric binding of the T7 lysozyme to T7 RNAP halts the RNA synthesis of T7 RNAP. The IZ-DS sequence was introduced to the T7 lysozyme. By optimizing the introduction site of the IZ-DS sequence in the T7 lysozyme, we succeeded in constructing the T7 lysozyme variant, DS-Lys²³. In the absence of IZ-3K, the mixture of T7 RNAP and DS-Lys²³ exhibited RNA synthesis due to the weakening of the interaction between T7 RNAP and DS-Lys²³. Whereas, after the addition of IZ-3K, RNA synthesis was significantly suppressed by the binding of DS-Lys²³/IZ-3K complex. The present methodology using a designed ligand-dependent metamorphosis protein sequence constitutes another possible method for the de novo manipulation of various functions of natural protein complexes.