Abstract
BackgroundDeath-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 FindingsIn 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/SignificanceThe 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.
Highlights
Death-Associated Protein Kinase (DAPK) was first identified in a functional screening assay that aimed at identifying genes involved in interferon-c induced cell death [1]
High-resolution crystallographic structures of various conformations of the DAPK catalytic domain [3,4,5,6] including the apo-form [4] and complexes containing bound nucleotides [4,5] or small molecule inhibitor fragments [3] have identified structures that are key to catalytic activity [7,8] as well as novel structural features [4,7,9] potentially involved in protein-protein interactions
This prior work with heterodimer formation led us to postulate that DAPK might self-associate to form homodimers under the appropriate experimental conditions, and that the basic loop region of DAPK might be involved in such self-association
Summary
Death-Associated Protein Kinase (DAPK) was first identified in a functional screening assay that aimed at identifying genes involved in interferon-c induced cell death [1]. The DAPK crystallographic structural studies [4] identified a basic loop that is not required for enzymatic activity [7], but has sequence homology to regions of other death protein kinases [4,7,9]. The basic loop region of ZIPK is considered key to functional heterodimer formation between ZIPK and DAPK catalytic domains [9]. 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
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