Purification, crystallization and preliminary X-ray diffraction analysis of the kinase domain of human tousled-like kinase 2
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Updated: May 2, 2026
Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
Published on: February 21, 2019
Ana M Garrote1, Pilar Redondo1, Guillermo Montoya1
1Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Abstract:
Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in chromatin dynamics, including DNA replication and repair, transcription and chromosome segregation. The two members of the family reported in humans, namely TLK1 and TLK2, localize to the cell nucleus and are capable of forming homo- or hetero-oligomers by themselves. To characterize the role of TLK2, its C-terminal kinase domain was cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4122 and cubic P213. The latter produced the best diffracting crystal (3.4 Å resolution using synchrotron radiation), with unit-cell parameters a = b = c = 126.05 Å, α = β = γ = 90°. The asymmetric unit contained one protein molecule, with a Matthews coefficient of 4.59 Å(3) Da(-1) and a solvent content of 73.23%.
Insights
Tousled-like kinase 2 (TLK2) is crucial for chromatin dynamics. Researchers crystallized the TLK2 kinase domain, enabling structural studies to understand its role in DNA replication and repair.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Tousled-like kinases (TLKs) are serine/threonine protein kinases.
- TLKs regulate chromatin dynamics, including DNA replication, repair, and transcription.
- Human TLK1 and TLK2 are nuclear proteins that can form oligomers.
Purpose of the Study:
- To characterize the role of Tousled-like kinase 2 (TLK2).
- To obtain structural information of the TLK2 kinase domain.
- To facilitate further studies on TLK2 function in chromatin dynamics.
Main Methods:
- Cloning and overexpression of the TLK2 C-terminal kinase domain in E. coli.
- Purification of the TLK2 kinase domain to homogeneity.
- Crystallization of TLK2 in the presence of ATP-γ-S.
- X-ray diffraction analysis of TLK2 crystals.
Main Results:
- Crystals of the TLK2 kinase domain were obtained in two space groups (I4122 and P213).
- The P213 space group yielded the best diffracting crystals (3.4 Å resolution).
- Unit-cell parameters and solvent content were determined for the P213 crystal.
Conclusions:
- The successful crystallization of the TLK2 kinase domain provides a basis for its structural determination.
- This structural information will be vital for understanding TLK2's mechanism in chromatin regulation.
- The study paves the way for future investigations into TLK2's role in cellular processes.
