Structure of the Dimeric Autoinhibited Conformation of DAPK2, a Pro-Apoptotic Protein Kinase

Abstract

The death-associated protein kinase (DAPK) family has been characterized as a group of pro-apoptotic serine/threonine kinases that share specific structural features in their catalytic kinase domain. Two of the DAPK family members, DAPK1 and DAPK2, are calmodulin-dependent protein kinases that are regulated by oligomerization, calmodulin binding, and autophosphorylation. In this study, we have determined the crystal and solution structures of murine DAPK2 in the presence of the autoinhibitory domain, with and without bound nucleotides in the active site. The crystal structure shows dimers of DAPK2 in a conformation that is not permissible for protein substrate binding. Two different conformations were seen in the active site upon the introduction of nucleotide ligands. The monomeric and dimeric forms of DAPK2 were further analyzed for solution structure, and the results indicate that the dimers of DAPK2 are indeed formed through the association of two apposed catalytic domains, as seen in the crystal structure. The structures can be further used to build a model for DAPK2 autophosphorylation and to compare with closely related kinases, of which especially DAPK1 is an actively studied drug target. Our structures also provide a model for both homodimerization and heterodimerization of the catalytic domain between members of the DAPK family. The fingerprint of the DAPK family, the basic loop, plays a central role in the dimerization of the kinase domain.

Perfluorooctane sulfonate induces apoptosis of hippocampal neurons in rat offspring associated with calcium overload

“…The alg-2 gene was discovered to be a pro-apoptotic calcium binding protein, and it has been proposed that it is involved in calcium mediated signaling processes to regulate cell death. 45,46 In the present research, alg-2 and dapk2 were over-expressed by PFOS. 45 In the present study, alg-2, dapk2 and [Ca 2+ ] i changed concurrently in a similar pattern, demonstrating again that PFOS-induced apoptosis is possibly related to the disturb- ance in calcium homeostasis.…”

Death-associated protein kinase 2: Regulator of apoptosis, autophagy and inflammation

“…At the N-terminus, ranging from residue 23 to 285, the enzyme contains its catalytic domain with 80% homology to DAPK1 kinase domain (Kawai et al, 1999). The crystal structure of the kinase domain was resolved a few years ago, suggesting that DAPK2 catalytic domains form dimers (Patel et al, 2011). This is in agreement with the identification of a key structural feature of the DAPK family of proteins, a short segment of mostly positively charged residues termed the basic loop (amino acids 55-66), which was shown to mediate DAPK homodimerization (Zimmermann et al, 2010).…”

“…The nature and impact of homodimerization on DAPK2 activity is a matter of controversy. On the one hand, the resolution of the crystal structure of DAPK2 catalytic domain revealed that homodimerization of DAPK2 mediated by binding of two apposed catalytic domains to each other does not allow for substrate binding (Patel et al, 2011), implying decreased DAPK2 activity upon homodimerzation. On the other hand, C-tail deletion mutants demonstrated that homodimerization mediated by the C-terminus increases DAPK2 activity and is necessary for the DAPK2-mediated membrane blebbing (Inbal et al, 2000(Inbal et al, , 2002Shani et al, 2001).…”

The many structural faces of calmodulin: a multitasking molecular jackknife

“…modes 1-11 (Majava and Kursula 2009), 1-16 (Osawa et al 1999), and 1-17 (Maximciuc et al 2006) have been identified in crystal structures. Furthermore, the complexes of CaM with peptides from DAP kinases have a combined 1-10-14 mode, where two hydrophobic residues from the peptide lie in the same pocket of CaM in the crystal structures (Kuczera and Kursula 2012; Patel et al 2011). Also, the complex with a myristoylated peptide from CAP-23/NAP-22 has a classical collapsed conformation, even though the ligand peptide does not form an α helix and the myristoyl tail also binds between the CaM lobes (Matsubara et al 2004).…”