Expression and purification of DYRK1A kinase domain in complex with its folding intermediate-selective inhibitor
Ninako Kimura1, Kanako Saito1, Takashi Niwa2
1Laboratory for Drug Target Research, Department of Agriculture, Graduate School of Science and Technology, Shinshu University, 8304 Minami-Minowa, Kami-Ina, Nagano, 399-4598, Japan.
Abstract:
The kinase DYRK1A phosphorylates substrate proteins that are involved in the progression of many diseases. DYRK1A also phosphorylates its own residues on key elements intramolecularly to activate and stabilize itself during the folding process. Once the folding process of DYRK1A has completed, it can no longer catalyzes the intramolecular reaction, suggesting that a transitional intermediate state that catalyzes the autophosphorylation exists. In the previous study, we identified a small molecule, designated as FINDY, that selectively inhibits the folding intermediate of DYRK1A. Although evidence has suggested that FINDY targets the ATP-binding pocket of DYRK1A, it remains elusive as to whether the DYRK1A kinase domain could be purified as a complex with FINDY. In this study, we successfully expressed and purified the kinase domain of DYRK1A in complex with FINDY. The DYRK1A kinase domain was expressed as a fusion protein with a hexahistidine tag and ZZ-domain (His-ZZ-DYRK1A) at 6 °C by using a cold shock induction system in Escherichia coli cells. The cells were incubated with FINDY. The cell pellets were gently extracted on ice and subjected to immobilized-metal affinity chromatography. The amount of FINDY in the elution fraction was measured by UV absorbance specific for FINDY. The eluate contained FINDY with the ratio of FINDY to DYRK1A protein being 0.15 in quadruplicate experiments. Thus, this study demonstrates the direct interaction between the DYRK1A kinase domain and FINDY, paving the way for structural determination of the complex.
Insights
We purified the DYRK1A kinase domain bound to the inhibitor FINDY. This confirms direct interaction and enables structural studies of this disease-related kinase inhibitor complex.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- DYRK1A kinase is crucial in disease progression.
- DYRK1A autophosphorylation during folding suggests a unique intermediate state.
- FINDY is a small molecule inhibitor targeting this intermediate.
Purpose of the Study:
- To confirm the direct interaction between the DYRK1A kinase domain and FINDY.
- To purify the DYRK1A-FINDY complex for further structural analysis.
Main Methods:
- Expressed DYRK1A kinase domain as a His-ZZ-DYRK1A fusion protein.
- Utilized a cold shock induction system in E. coli.
- Purified the complex using immobilized-metal affinity chromatography.
Main Results:
- Successfully expressed and purified the DYRK1A kinase domain in complex with FINDY.
- Quantified FINDY bound to DYRK1A, with a ratio of 0.15.
- Demonstrated direct physical interaction between the kinase and the inhibitor.
Conclusions:
- This study validates the direct binding of FINDY to the DYRK1A kinase domain.
- The purified complex provides a foundation for future structural determination.
- This work advances the development of DYRK1A-targeted therapeutics.
