Wang L and Zhou M (2023), Structure of a eukaryotic cholinephosphotransfe...

XB-ART-59803

Nat Commun 2023 May 13;141:2753. doi: 10.1038/s41467-023-38003-9.

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Structure of a eukaryotic cholinephosphotransferase-1 reveals mechanisms of substrate recognition and catalysis.

Wang L , Zhou M .

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Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic cell membranes. In eukaryotes, two highly homologous enzymes, cholinephosphotransferase-1 (CHPT1) and choline/ethanolamine phosphotransferase-1 (CEPT1) catalyze the final step of de novo PC synthesis. CHPT1/CEPT1 joins two substrates, cytidine diphosphate-choline (CDP-choline) and diacylglycerol (DAG), to produce PC, and Mg2+ is required for the reaction. However, mechanisms of substrate recognition and catalysis remain unresolved. Here we report structures of a CHPT1 from Xenopus laevis (xlCHPT1) determined by cryo-electron microscopy to an overall resolution of ~3.2 Å. xlCHPT1 forms a homodimer, and each protomer has 10 transmembrane helices (TMs). The first 6 TMs carve out a cone-shaped enclosure in the membrane in which the catalysis occurs. The enclosure opens to the cytosolic side, where a CDP-choline and two Mg2+ are coordinated. The structures identify a catalytic site unique to eukaryotic CHPT1/CEPT1 and suggest an entryway for DAG. The structures also reveal an internal pseudo two-fold symmetry between TM3-6 and TM7-10, and suggest that CHPT1/CEPT1 may have evolved from their distant prokaryotic ancestors through gene duplication.

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Genes referenced: pycard tpm3

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	Fig. 1. Function of purified xlCHPT1.a–b Initial rate of reaction in different concentrations of CDP-choline (a) or CDP-ethanolamine (b). c Relative activity of xlCHPT1 in the presence of different cations. Each symbol or bar is the average of three independent measurements, and error bars are standard errors of the mean (s.e.m.). The solid lines in a, b are fit of the data point to a Michaelis–Menten equation.
	Fig. 2. Overall structure of xlCHPT1.a Density map of dimeric xlCHPT1. Density of the two monomers is colored in cyan and pink, and the density of lipids is colored in yellow. b Model of dimeric xlCHPT1. Two monomers are shown as cartoons in cyan and pink, and substrates and lipids are shown as yellow sticks. Two Mg2+ are shown as green spheres. c Structure of xlCHPT1 monomer in two orientations. d Membrane topology plot of an xlCHPT1 monomer.
	Fig. 3. Mg2+ and CDP-choline-binding sites.a Overview of CDP-choline and Mg2+-binding sites in two orientations. xlCHPT1 is shown as cartoon, CDP-choline as a stick, and Mg2+ as green spheres. b–c Density maps of CDP or CDP-choline with Mg2+. Density map is shown as transparent blue surface, Mg2+ as spheres, and CDP or CDP-choline as sticks. d–f Coordination of Mg2+, CDP, or CDP-choline in xlCHPT1. Binding site residues and CDP/CDP-choline are both shown as sticks but with a different color scheme. g Relative activity of wide type and mutants of xlCHPT1. Each symbol is an average of three independent measurements. Error bars are standard errors of the mean (s.e.m.). h The catalytic site, which is marked as a star, and the proposed catalytic residues, E129 and H133, are shown as sticks.
	Fig. 4. Dimerization interface.a–b Dimeric xlCHPT1 structure is shown as cartoon in two orientations, and lipids are shown as yellow sticks. The green and orange boxes mark the region with close-up views shown in c & d. c Residues at the dimer interface. Two monomers are shown in cyan and pink. d Lipids trapped at the dimer interface. Density of lipids is shown as a transparent gray surface, and partially resolved lipid molecules are shown as sticks.
	Fig. 5. Substrate entry and reaction mechanism.a–b Proposed entry pathways for both substrates. CDP-choline and DAG are shown as stick and spheres, respectively. xlCHPT1 is shown as cartoon. Proposed active site residues H133 and E129 are shown as spheres. c Proposed catalytic mechanism of CHPT1. E129 and H133 activate the 3-hydroxyl on DAG to enhance nucleophilic attack on the phosphate group of CDP-choline.

References [+] :

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed