Kumpei Yamanishi, Wataru Kumano, Shin-Ichi Terawaki, Yoshiki Higuchi and Naoki Shibata* Pages 1 - 6 ( 6 )
Background: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/β-catenin signalling pathway, but this interaction mechanism is still unknown.
Objective: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation.
Method: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them.
Results: The crystal of the DAX–DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wnt-signalosome model.
Conclusion: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.
Wnt signaling pathway, Dishevelled, Axin, DIX domain, protein complex, crystallization, X-ray diffraction analysis
Graduate School of Life Science, University of Hyogo, Ako-gun, Graduate School of Life Science, University of Hyogo, Ako-gun, Graduate School of Science and Technology, Gunma University, Kiryu, Graduate School of Life Science, University of Hyogo, Ako-gun, Graduate School of Life Science, University of Hyogo, Ako-gun