Head-to-Tail Complex of Dishevelled and Axin-DIX Domains: Expression, Purification, Crystallographic Studies and Packing Analysis

Protein Pept Lett. 2019;26(10):792-797. doi: 10.2174/0929866526666190425152721.

Abstract

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.

Methods: 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 Wntsignalosome 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.

Keywords: DIX domain; Wnt signaling pathway; X-ray diffraction analysis; axin; crystallisation; dishevelled; protein complex..

MeSH terms

  • Amino Acid Sequence
  • Axin Protein / chemistry*
  • Axin Protein / genetics*
  • Crystallization
  • Crystallography, X-Ray / methods
  • Dishevelled Proteins / chemistry*
  • Dishevelled Proteins / genetics
  • Escherichia coli
  • Gene Expression Regulation
  • Humans
  • Protein Binding
  • Protein Domains / genetics*
  • Wnt Signaling Pathway

Substances

  • Axin Protein
  • Dishevelled Proteins