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

Graphical abstract
Highlights • We revealed the intra-molecular interaction of DGKα. • The EF-hand motifs of DGKα directly binds to its C1 domains. • The intra-molecular interaction was negatively regulated by Ca2+. • The intra-molecular interaction is important for the activation mechanism of DGKα.
Diacylglycerol kinase (DGK) α, which is activated by Ca2+, contains a recoverin homology (RVH) domain, tandem repeats of two Ca2+-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. In this study, we narrowed down the interaction regions of DGKα. 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 Ca2+. These results indicate that the Ca2+-induced dissociation of the intra-molecular interaction between the EF-hand motifs and the C1 domains of DGKα is the key event that regulates the activity of the enzyme.