Exploring the AlphaFold Predicted Conformational Properties of Human Diacylglycerol Kinases.

Diacylglycerol kinases (DGKs) are important enzymes in molecular membrane biology, as they can lower the concentration of diacylglycerol through phosphorylation while at the same time producing phosphatidic acid. Dysfunction of DGK is linked with multiple diseases including cancer and autoimmune disorders. Currently, the high-resolution structures have not been determined for any of the 10 human DGK paralogs, which has made it difficult to gain a more complete understanding of the enzyme's mechanism of action and regulation. In the present study, we have taken advantage of the significant developments in protein structural prediction technology by artificial intelligence (i.e., Alphafold 2.0), to conduct a comprehensive investigation on the properties of all 10 human DGK paralogs. Structural alignment of the predictions reveals that the C1, catalytic, and accessory domains are conserved in their spatial arrangement relative to each other, across all paralogs. This suggests a critical role played by this domain architecture in DGK function. Moreover, docking studies corroborate the existence of a conserved ATP-binding site between the catalytic and accessory domains. Interestingly, the ATP bound to the interdomain cleft was also found to be in proximity of the conserved glycine-rich motif, which in protein kinases has been suggested to function in ATP binding. Lastly, the spatial arrangement of DGK, with respect to the membrane, reveals that most paralogs possess a more energetically favorable interaction with curved membranes. In conclusion, AlphaFold predictions of human DGKs provide novel insights into the enzyme's structural and functional properties while also paving the way for future experimentation.

Human Diacylglycerol Kinase ε N-Terminal Segment Regulates the Phosphatidylinositol Cycle, Controlling the Rate but Not the Acyl Chain Composition of Its Lipid Intermediates.

Diacylglycerol kinase ε (DGKε), an enzyme of the phosphatidylinositol (PI) cycle, bears a highly conserved hydrophobic N-terminal segment, which was proposed to anchor the enzyme into the membrane. However, the importance of this segment to the DGKε function remains to be determined. To address this question, it is here reported an in silico and in vitro combined research strategy. Capitalizing on the AlphaFold 2.0 predicted structure of human DGKε, it is shown that its hydrophobic N-terminal segment anchors it into the membrane via a transmembrane α-helix. Coarse-grained based elastic network model studies showed that a conformational change in the hydrophobic N-terminal segment determines the proximity between the active site of DGKε and the membrane-water interface, likely regulating its kinase activity. In vitro studies with a purified DGKε construct lacking the hydrophobic N-terminal segment (His-SUMO*-Δ50-DGKε) corroborated the role of the N-terminus in regulating DGKε enzymatic properties. The comparison between the enzymatic properties of DGKε and His-SUMO*-Δ50-DGKε showed that the conserved N-terminal segment markedly inhibits the enzyme activity and its sensitivity to membrane intrinsic negative curvature, while also playing a role in the modulation of the enzyme by phosphatidylserine. On the other hand, this segment did not strongly affect its diacylglycerol acyl chain specificity, the modulation of the enzyme by membrane morphological changes, or the activation by phosphatidic acid-rich lipid domains. Hence, these results suggest that the conservation of the hydrophobic N-terminal segment of DGKε throughout evolution guaranteed not only membrane anchorage but also an efficient and elegant manner to regulate the rate of the PI cycle.

Membrane shape as determinant of protein properties.

Membrane lipids play a role in the modulation of a variety of biological processes. This is often achieved through fine-tuned changes in membrane physical and chemical properties. While some membrane physical properties (e.g., curvature, lipid domains, fluidity) have received increased scientific attention over the years, only recently has membrane shape emerged as an active modulator of protein properties. Biological membranes are mostly found organized into a lipid bilayer arrangement, in which the spontaneous shape is an intrinsically flat, planar morphology (in relation to the size of proteins). However, it is known that many cells and organelles have non-planar morphologies. In addition, perturbations in membrane morphology occur in a variety of biological processes. Recent studies have shown that membrane shape can modulate a variety of biological processes by determining protein properties. While membrane shape generation modulates proteins via changes in membrane mechanical properties, membrane shape recognition regulates proteins by providing the optimal surface for interaction. Hence, membranes have evolved an elegant mechanism to couple mesoscopic perturbations to molecular properties and vice-versa. In this review, the regulation of the enzymatic properties of two isoforms of mammalian diacylglycerol kinase, which play important roles in cellular signal transductions, will be used to exemplify the recent advancements in the field of membrane shape recognition, as well as future challenges and perspectives.