Molecular Determinants for Interfacial Binding and Conformational Change in a Soluble Diacylglycerol Kinase

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. Lys 15 and Lys 165 were required for DgkB to dock with PtdGro vesicles and flank the entrance to the DgkB active site. Mg 2؉ was required for vesicle binding. The compromised vesicle binding by mutants in the key asparate residues forming the structural Mg 2؉ -aspartatewater network within the substrate binding domain revealed that interfacial binding of DgkB required a Mg 2؉ -dependent conformational change. DgkB interaction with phospholipid vesicles was not influenced by the presence of ATP, but anionic vesicles decreased the K m of the enzyme for ATP. Arg 100 and Lys 15 are two surface residues in the ATP binding domain that were necessary for high affinity ATP binding. The key residues responsible for the structural Mg 2؉ 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.The diacylglycerol (DAG) 2 kinase superfamily (Pfam00781) defines a group of soluble lipid kinases that phosphorylate membrane-associated DAG and function in regulating intracellular phospholipid metabolism and signaling. The mammalian family members share a conserved catalytic core domain that is associated with a variety of modules that confer intracellular membrane targeting specificity (for reviews, see Refs. 1 and 2). Staphylococcus aureus DgkB is a prototypical member of Pfam00781 that has the catalytic core of the DAG kinase superfamily, but lacks the ancillary domains characteristic of its mammalian cousins. DgkB proteins are essential in Gram-positive bacteria to re-introduce the large amount of DAG arising from lipoteichoic acid biosynthesis into the phospholipid biosynthetic pathway (3). DAG is exclusively found in the membrane phospholipid bilayer and the soluble DgkB must be capable of interfacial catalysis despite the absence of the membrane targeting modules present in its mammalian counterparts.Interfacial activation is a key feature of soluble proteins that act on substrates embedded in a phospholipid bilayer and has been extensively studied in phospholipid hydrolases (4, 5). These enzymes can utilize monomeric substrates, but their activity increases significantly when substrate is presented in micelles or unilamellar vesicles. Electrostatic effects are crucial to interfacial recognition by the soluble phospholipase A 2 proteins (4), and the structu...

Phosphatidic acid synthesis in bacteria

“…The DgkB surface is overall electronegative, except for an electropositive patch consisting of Lys15, Arg20, and Lys165 adjacent to the active site entrance. DgkB binds to anionic phospholipid vesicles with high affinity, and analysis of the K15A, R20A, and K165A mutant proteins confirm the role of these residues in interfacial docking [99]. Binding of DgkB to anionic phospholipids lowers the K m for ATP from 3.7 mM to 32 μM illustrating that interfacial binding triggers a conformational change that activates the kinase.…”

Bacterial lipids: Metabolism and membrane homeostasis

“…This soluble DAG kinase is highly specific for the phosphorylation of DAG over other phospholipids, and its primary sequence places it in the same protein family as the mammalian signaling DAG kinases. Unlike the integral membrane DgkA, DgkB is an interfacial enzyme that uses positively charged lysine residues on its surface to dock on the anionic surface of the cell membrane [138]. This property is demonstrated in vitro through enzymatic assays showing a robust increase in DgkB activity when DAG is incorporated into negatively-charged PtdGro vesicles, as opposed to neutrally charged bilayers [138].…”

“…Unlike the integral membrane DgkA, DgkB is an interfacial enzyme that uses positively charged lysine residues on its surface to dock on the anionic surface of the cell membrane [138]. This property is demonstrated in vitro through enzymatic assays showing a robust increase in DgkB activity when DAG is incorporated into negatively-charged PtdGro vesicles, as opposed to neutrally charged bilayers [138]. Complementation studies of a dgkB mutant with an inducible plasmid expressing dgkB revealed the gene to be essential in B. subtilis unless LTA synthesis is disrupted by mutations in yflE, a gene homologous to ltaS of S. aureus.…”

Development of a Novel β-Secretase Binding Assay Using the AlphaScreen Platform

“…The enhanced sensitivity (~10×) that we observed is consistent with what was reported using the AlphaScreen platform compared with other assay formats for other targets as well. 31,32 The donor and acceptor beads contain multiple binding sites, and thus the enhanced sensitivity of this platform is attributed to a bead avidity effect. The bead avidity effect allows for weak interactions (in the 50-100 µM range) to be successfully measured using this format.…”