Abstract
DgkB is a soluble diacylglycerol (DAG) kinase that is essential for membrane lipid homeostasis in many Gram-positive pathogens. Anionic phospholipids, like phosphatidylglycerol (PtdGro), were required for DgkB to recognize diacylglycerol embedded in a phospholipid bilayer. An activity-independent vesicle binding assay was used to determine the role of specific residues in DgkB-PtdGro interactions. Lys15 and Lys165 were required for DgkB to dock with PtdGro vesicles and flank the entrance to the DgkB active site. Mg2+ was required for vesicle binding. The compromised vesicle binding by mutants in the key asparate residues forming the structural Mg2+-aspartate-water network within the substrate binding domain revealed that interfacial binding of DgkB required a Mg2+-dependent conformational change. DgkB interaction with phospholipid vesicles was not influenced by the presence of ATP, but anionic vesicles decreased the Km of the enzyme for ATP. Arg100 and Lys15 are two surface residues in the ATP binding domain that were necessary for high affinity ATP binding. The key residues responsible for the structural Mg2+ binding site, the conformational changes that increase ATP affinity, and interfacial recognition of anionic phospholipids were identical in DgkB and the mammalian diacylglycerol kinase catalytic cores. This sequence conservation suggests that the mammalian enzymes also require a structural divalent cation and surface positively charged residues to bind phospholipid bilayers and trigger conformational changes that accelerate catalysis.
Highlights
IntroductionPhospholipases A and C do undergo subtle conformational changes upon binding to the interface that cooperate in promoting bilayer association and processive catalysis [5, 7, 15,16,17,18]
This structural analysis suggests that docking of DgkB to a phospholipid bilayer via these surface features would orient the protein with the active site entrance facing the phospholipid bilayer
The catalytic center of DgkB has an incompletely formed ATP binding site indicating that a conformational change to high-affinity ATP binding occurs upon docking of the protein to the bilayer
Summary
Phospholipases A and C do undergo subtle conformational changes upon binding to the interface that cooperate in promoting bilayer association and processive catalysis [5, 7, 15,16,17,18]. The goals of this study are to define the i-face of DgkB, to identify the molecular determinants on its surface that are required for interfacial binding, and to characterize the conformational changes associated with the activation of enzyme. We predict that these findings will extend to the entire superfamily of soluble DAG kinases
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