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

Abstract

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.

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

“…As seen with other eukaryotic CDP-APs 24,25 , the TM helices that form the catalytic motif of yCPT1 superimpose onto the six TM helices found in prokaryotic CDP-APs ( e.g. , r.m.s.d.…”

“…Here we present the structures of full-length yCPT1 in different substrate-bound states determined by single particle cryo-electron microscopy (cryo-EM). These structures show that, like other CDP-APs, yCPT1 forms a dimer although using an entirely different interface than seen in the known prokaryotic or eukaryotic structures 17–25 . Our structures of yCPT1 in complex with either DAG or CDP-choline delineate the binding mode of these substrates and mutational analyses identified the residues critical for the DAG acyl-chain preference and head-group selectivity.…”

“…These structures provided valuable insights into the overall architecture, molecular basis of substrate recognition, and the catalytic mechanism of this family of integral membrane enzymes. Recently, two structures of eukaryotic CDP-APs, human CEPT1 and Xenopus laevis CHPT1, were determined 24,25 . The structures showed the overall architecture of each enzyme, the binding mode of PC, and suggested possible substrate entry sites.…”

“…Metal ions, such as Ca 2+ , Zn 2+ , Mn 2+ , and Na + , are coordinated with the four aspartate residues in the signature motif of various prokaryotic CDP-APs. Because yCPT1, like xlCHPT1 and hCHPT1, requires Mg 2+ or Mn 2+ for activity 25,28,29 and we included 10mM MgCl 2 in the sample buffer, we modeled Mg 2+ into the densities observed at the catalytic center of each protomer (Supplementary Fig. 4b).…”

The small molecule CBR-5884 inhibits theCandida albicansphosphatidylserine synthase

“…Besides the characterization of the substrate-binding residues, the C. albicans Cho1 has also been solubilized and purified as a hexameric protein (33), distinct from all the CDP-AP enzymes with solved structures (30,32,(34)(35)(36)(37)(38)(39)(40), which are dimers. The hexameric C. albicans Cho1 can be separated into a trimer of stable dimers, indicating the hexamer might be [i] an early oligomer state, since Cho1 was solubilized from the early-to-mid log phase of C. albicans or [ii] species-specific (33).…”