Dataset Information

EF-hand motifs of diacylglycerol kinase ? interact intra-molecularly with its C1 domains.

ABSTRACT: Diacylglycerol kinase (DGK) ?, which is activated by Ca(2+), contains a recoverin homology (RVH) domain, tandem repeats of two Ca(2+)-binding EF-hand motifs, two cysteine-rich C1 domains and the catalytic domain. We previously found that a DGK? mutant lacking the RVH domain and EF-hands was constitutively active and that the N-terminal region of DGK?, consisting of the RVH domain and EF-hand motifs, interacted intra-molecularly with the C-terminal region containing the C1 and catalytic domains. In this study, we narrowed down the interaction regions of DGK?. At the C-terminal region, the C1 domains are responsible for the intra-molecular interaction. At the N-terminal region, the EF-hand motifs mainly contribute to the interaction. Moreover, using highly purified EF-hand motifs and C1 domains, we demonstrate that they directly bind to each other. The co-precipitation of these two domains was clearly attenuated by the addition of Ca(2+). These results indicate that the Ca(2+)-induced dissociation of the intra-molecular interaction between the EF-hand motifs and the C1 domains of DGK? is the key event that regulates the activity of the enzyme.

SUBMITTER: Yamamoto T

PROVIDER: S-EPMC4050180 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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EF-hand motifs of diacylglycerol kinase α interact intra-molecularly with its C1 domains.

Yamamoto Tatsuya T Sakai Hiromichi H Sakane Fumio F

FEBS open bio 20140418

Diacylglycerol kinase (DGK) α, which is activated by Ca(2+), contains a recoverin homology (RVH) domain, tandem repeats of two Ca(2+)-binding EF-hand motifs, two cysteine-rich C1 domains and the catalytic domain. We previously found that a DGKα mutant lacking the RVH domain and EF-hands was constitutively active and that the N-terminal region of DGKα, consisting of the RVH domain and EF-hand motifs, interacted intra-molecularly with the C-terminal region containing the C1 and catalytic domains. ...[more]

PMID: 24918053

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Project description:Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that phosphorylate diacylglycerol into phosphatidic acid, modulating the levels of these key signaling lipids. Recently, increasing attention has been paid to DGKα isozyme as a potential target for cancer immunotherapy. We have previously shown that DGKα is positively regulated by Ca2+ binding to its N-terminal EF-hand domains (DGKα-EF). However, little progress has been made for the structural biology of mammalian DGKs and the molecular mechanism underlying the Ca2+ -triggered activation remains unclear. Here we report the first crystal structure of Ca2+ -bound DGKα-EF and analyze the structural changes upon binding to Ca2+ . DGKα-EF adopts a canonical EF-hand fold, but unexpectedly, has an additional α-helix (often called a ligand mimic [LM] helix), which is packed into the hydrophobic core. Biophysical and biochemical analyses reveal that DGKα-EF adopts a protease-susceptible "open" conformation without Ca2+ that tends to form a dimer. Cooperative binding of two Ca2+ ions dissociates the dimer into a well-folded monomer, which resists to proteolysis. Taken together, our results provide experimental evidence that Ca2+ binding induces substantial conformational changes in DGKα-EF, which likely regulates intra-molecular interactions responsible for the activation of DGKα and suggest a possible role of the LM helix for the Ca2+ -induced conformational changes. SIGNIFICANCE STATEMENT: Diacylglycerol kinases (DGKs), which modulates the levels of two lipid second messengers, diacylglycerol and phosphatidic acid, is still structurally enigmatic enzymes since its first identification in 1959. We here present the first crystal structure of EF-hand domains of diacylglycerol kinase α in its Ca2+ bound form and characterize Ca2+ -induced conformational changes, which likely regulates intra-molecular interactions. Our study paves the way for future studies to understand the structural basis of DGK isozymes.

Dataset Information

EF-hand motifs of diacylglycerol kinase ? interact intra-molecularly with its C1 domains.

Publications

EF-hand motifs of diacylglycerol kinase α interact intra-molecularly with its C1 domains.

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