Structural basis for catalysis and selectivity of phospholipid synthesis by eukaryotic choline-phosphotransferase

Abstract

Phospholipids are the most abundant component in lipid membranes and are essential for the structural and functional integrity of the cell. In eukaryotic cells, phospholipids are primarily synthesized de novo through the Kennedy pathway that involves multiple enzymatic processes. The terminal reaction is mediated by a group of cytidine-5'-diphosphate (CDP)-choline /CDP-ethanolamine-phosphotransferases (CPT/EPT) that use 1,2-diacylglycerol (DAG) and CDP-choline or CDP-ethanolamine to produce phosphatidylcholine (PC) or phosphatidylethanolamine (PE) that are the main phospholipids in eukaryotic cells. Here we present the structure of the yeast CPT1 in multiple substrate-bound states. Structural and functional analysis of these binding-sites reveal the critical residues for the DAG acyl-chain preference and the choline/ethanolamine selectivity. Additionally, we present the structure in complex with a potent inhibitor characterized in this study. The ensemble of structures allows us to propose the reaction mechanism for phospholipid biosynthesis by the family of CDP-alcohol phosphotransferases (CDP-APs).

Fig. 1. Overall architecture of yCPT1.

a 3.2 Å resolution cryo-EM density map of dimeric yCPT1. Each ligand, motif, and assembly are colored according to the panel legend: yellow, catalytic assembly; purple, CDP-AP signature motif; gray, dimerization assembly; brown, N-terminal stretch; cyan, phosphatidylcholine (PC); blue, diacylglycerol (DAG). Positions of lumenal and cytoplasmic membranes shown in top panel. b Model of dimeric yCPT1. Atomic models corresponding to the density in panel a. In one protomer of the model each ligand, motif, and assembly are colored similarly as in panel a. In the lumenal view, the TM helix is numbered and the juxtamembrane helix (JMH) is labeled. Inter-monomer PCs and DAG are depicted as sticks. c Surface model of yCPT1 colored with hydrophobicity index by Chimera. DAG is shown as balls within the surface model colored in blue(carbon) and red(oxygen). The cross-section plane depicted in panel d is also shown. d Cross-section of yCPT1 at the substrate-binding pocket. CDP-choline-binding site is enclosed in a solid red line. The color code of the models and the hydrophobicity index of yCPT1 is the same as shown in the panel c.

Fig. 2. DAG binding mode of yCPT1.

a Model of yCPT1 protomer as viewed from the membrane plane. The box highlights the position of the DAG-binding site and is shown expanded in panels b and c. The cryo-EM density of DAG is shown in blue with the model also shown in blue. Acyl-chains from DAG occupy the hydrophobic spaces inside of yCPT1. b Enlarged view of DAG-binding site. Each of the two acyl-chains of DAG occupies two distinct hydrophobic channels: Channel 1, the channel between TM4 and TM6; Channel 2, the channel between TM5 and TM6. c The same DAG-binding site is viewed from the lumenal side. Note that Phe146 shown as sticks is located at the exit site of channel 1. d DAG-surrounding residues in yCPT1. LigPlot2 is used for the analysis and the illustration. Phe146 is shown in coral. e, f Acyl-chain preference of yCPT1 WT (blue circles) and yCPT1 F146L (coral triangles). Choline phosphotransferase activity was determined by quantifying the level of CMP released from CDP-choline (1 mM) using DAG 16:0-16:0 or DAG 18:1-18:1 at the indicated concentration. g The preference for C18-DAG was determined by dividing the amount of CMP released with PC 18:1-18:1 (300 μM) by that of CMP with PC 16:0-16:0 (300 μM). Data are presented as the mean ± SD from three independent measurements. Statistical significance was determined by unpaired two-sided t-test. Source data are provided as a Source Data file.

Fig. 3. CDP-choline and PC binding mode.

a, b 2.9 Å resolution cryo-EM density map of yCPT1 dimer bound with CDP-choline and PC. Density is colored in the same way as in Fig. 1: yellow, catalytic domain; purple, CDP-AP signature motif; gray, dimerization domain; brown, N-terminal stretch; cyan, phospholipid (PC) at the dimer interface: red, CDP-choline; salmon, PC in the substrate-binding site. The CDP-choline-binding pocket and the PC binding pocket are enlarged in zoom-in views showing stick models of these substrates. Part of CDP-choline is held in place by the CDP-binding motif (purple) located between TM helices 2 and 3. Two magnesium ions are present in the same place as DAG-bound state and depicted as green balls. c PC binding mode analyzed by LigPlot2. His118, that exhibits alternate conformations, is colored in purple. The residue with polar interaction with PC (Ser158) is shown as ball and sticks and labeled in green. d CDP-choline-binding mode viewed from the lumenal side. e CDP-choline-binding residues analyzed by LigPlot2. The residues with polar interaction with CDP-choline are shown as ball and sticks and labeled in green. f Displacement of the conserved tryptophan residue in the juxtamembrane helix (JMH) comparing the cryo-EM structure of yCPT1 (pale red) and the AF2 model of yEPT1 (turquoise). A conserved tryptophan (Trp35) is facing either alanine or glycine, respectively. g Time course for the PE synthesis by yCPT1 WT (blue circles) and yCPT1 A97G (red squares). h Preference of yCPT1 WT and yCPT1 A97G for CDP-ethanolamine. Ethanolamine phosphotransferase activity was assessed by quantification of deuterium-labeled PE synthesized from deuterium-labeled DAG (100 μM) and CDP-ethanolamine at the indicated concentrations. i Preference of yCPT1 WT and yCPT1 A97G for CDP-choline. Choline phosphotransferase activity was determined by quantifying CMP released from CDP-choline at the indicated concentration in the presence of DAG 16:0-16:0 (100 μM). Data are presented as the mean ± SD from three independent measurements. Statistical significance was determined by unpaired two-sided t-test. Source data are provided as a Source Data file.

Fig. 4. Chelerythrine is a yCPT1 inhibitor.

a Titration of chelerythrine for the inhibition of yCPT1 WT. The level of CMP released from CDP-choline was evaluated in the presence of chelerythrine at the indicated concentration. Data are presented as the mean ± SD from three independent measurements. b, c 3.2 Å resolution cryo-EM density map and model of yCPT1 dimer bound to chelerythrine. Density and model are depicted and colored in the same way as shown in Fig. 1: yellow, catalytic domain; purple, CDP-AP signature motif; gray, dimerization domain; brown, N-terminal stretch; cyan, phosphatidylcholine (PC). Chelerythrine is colored pale green. d The chelerythrine-binding pocket in yCPT1 is enlarged with chelerythrine and surrounding residues shown as a stick models. e LigPlot2 is used for the analysis of the chelerythrine-binding pocket and the illustration. Source data are provided as a Source Data file.

Fig. 5. Rotameric Histidine switch and proposed catalytic mechanism of yCPT1.

a Structural organization and the cryo-EM density map of the rotameric His118 conformations, water molecule, Glu114, Asp121, and CDP-choline. CDP-choline, Glu114, Asp121, and His118 are depicted as sticks and water molecule is shown as a red ball. b Validation of the critical roles of His118 and Glu114 in the catalytic activity of yCPT1. Choline phosphotransferase activity of yCPT1 WT (blue circles), yCPT1 E114A (green triangles) and yCPT1 H118A (red squares) was assessed by measuring the amount of CMP released from CDP-choline at the indicated concentration in the presence of DAG 16:0-16:0 (100 μM). Data are presented as the mean ± SD from three independent measurements. Statistical significance was determined using a one-way ANOVA with Tukey’s post hoc test. c (1) In the apo-state, His118 mostly adopts an “out” conformation pointing away from the catalytic center. (2) Upon the binding of DAG, the equilibrium of His118 moves toward “in” conformation closer to the catalytic center. (3) The binding of CDP-choline further stabilizes the equilibrium of His118 to the “in” position (4) His118 at the “in” position deprotonates 3-hydroxyl of DAG to trigger nucleophilic substitution reaction toward the phosphate group of CDP-choline. (5) End-products PC and CMP are released from their binding sites and yCPT1 returns to the apo-state. Source data are provided as a Source Data file.