Homodimerization of the death-associated protein kinase catalytic domain: development of a new small molecule fluorescent reporter
Related Experiment Video
Updated: Jun 6, 2026
Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation
Published on: March 5, 2018
Michael Zimmermann1, Cédric Atmanene, Qingyan Xu
1Laboratoire d'Innovation Thérapeutique, Unité Mixte de Recherche 7200, Centre National de la Recherche Scientifique, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Background:
Death-Associated Protein Kinase (DAPK) is a member of the Ca2+/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases.
Methodology/Principal Findings:
In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution.
Conclusions/Significance:
The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure.
Insights
Death-Associated Protein Kinase (DAPK) homodimerization involves its basic loop structure. This study reveals a monomer-dimer equilibrium critical for DAPK function and potential therapeutic targeting.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Death-Associated Protein Kinase (DAPK) is a Ca2+/calmodulin-regulated serine/threonine kinase.
- DAPK plays a role in induced cell death.
- DAPK is a therapeutic target for brain injuries and neurodegenerative diseases.
Purpose of the Study:
- To investigate the homodimerization of the DAPK catalytic domain.
- To elucidate the role of the basic loop structure in DAPK oligomerization.
- To develop methods for monitoring DAPK oligomerization state.
Main Methods:
- Nanoelectrospray ionization mass spectrometry (NSI-MS) was used to study DAPK catalytic domain and a basic loop mutant.
- Chemical biology approaches were employed to identify a fluorescent probe.
- The probe was used to monitor the protein's oligomerization state in solution.
Main Results:
- Homodimerization of the DAPK catalytic domain was observed.
- The basic loop structure was identified as crucial for DAPK homodimerization.
- A monomer-dimer equilibrium was elucidated, involving the basic loop.
Conclusions:
- A monomer-dimer equilibrium of DAPK was characterized.
- The basic loop is essential for DAPK homodimerization.
- An allosteric conformational change, independent of the basic loop, was detected using a fluorescent probe.
