Issue 15, 2009

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

Abstract

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 metamorphosisIZ-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.

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

Article information

Article type
Paper
Submitted
19 Jan 2009
Accepted
15 May 2009
First published
18 Jun 2009

Org. Biomol. Chem., 2009,7, 3102-3111

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

T. Mizuno, K. Suzuki, T. Imai, Y. Kitade, Y. Furutani, M. Kudou, M. Oda, H. Kandori, K. Tsumoto and T. Tanaka, Org. Biomol. Chem., 2009, 7, 3102 DOI: 10.1039/B901118H

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