Ca²⁺-induced structural changes and intramolecular interactions in N-terminal region of diacylglycerol kinase alpha

Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that modulate the levels of lipid messengers, diacylglycerol, and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. However, little progress has been made on the structural biology of DGKs, and a detailed understanding of the Ca²⁺-triggered activation of DGKα, for which the N-terminal domains likely play a critical role, remains unclear. We have recently shown that Ca²⁺ binding to DGKα-EF induces conformational changes from a protease-susceptible “open” conformation in the apo state to a well-folded one in its holo state. Here, we further studied the structural properties of DGKα N-terminal (RVH and EF) domains using a series of biophysical techniques. We first revealed that the N-terminal RVH domain is a novel Ca²⁺-binding domain, but the Ca²⁺-induced conformational changes mainly occur in the EF domain. This was corroborated by NMR experiments showing that the EF domain adopts a molten-globule like structure in the apo state. Further analyses using SEC-SAXS and NMR indicate that the partially unfolded EF domain interacts with RVH domain, likely via hydrophobic interactions in the absence of Ca²⁺, and this interaction is modified in the presence of Ca²⁺. Taken together, these results present novel insights into the structural rearrangement of DGKα N-terminal domains upon binding to Ca²⁺, which is essential for the activation of the enzyme.