Ca2+-stimulated translocation of cytosolic phospholipase A2α (cPLA2α) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLA2α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report cPLA2α C2-domain structure (2.2Å resolution) containing bound 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and Ca2+ ions. Two Ca2+ are complexed at locations previously reported for lipid-free C2-domain. One of these Ca2+ along with a third Ca2+ bridge the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid headgroup recognition via cation-π interaction with the PC trimethylammonium group. Mutagenesis analyses confirm Tyr96 and Asn65 function in PC binding selectivity by C2-domain and regulation of cPLA2α activity. The differing DHPC-binding mode of cPLA2α C2-domain, compared to phosphatidylserine or phosphatidylinositol 4,5-bisphosphate binding by other C2-domains, expands and deepens knowledge of lipid-binding mechanisms mediated by C2-domains.
Diffraction data have been deposited in PDB under the accession code 6IEJ
Structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2αProtein Data Bank, 6IEJ.
- Rhoderick E Brown
- Yoshinori Hirano
- Charles E Chalfant
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Arun Radhakrishnan, University of Texas Southwestern Medical Center, United States
© 2019, Hirano et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) is associated with human disorders, including Arts syndrome, retinal dystrophy, and gouty arthritis. Recent studies have demonstrated that PRPS can form filamentous cytoophidia in eukaryotes. Here, we show that PRPS forms cytoophidia in prokaryotes both in vitro and in vivo. Moreover, we solve two distinct filament structures of E. coli PRPS at near-atomic resolution using Cryo-EM. The formation of the two types of filaments is controlled by the binding of different ligands. One filament type is resistant to allosteric inhibition. The structural comparison reveals conformational changes of a regulatory flexible loop, which may regulate the binding of the allosteric inhibitor and the substrate ATP. A noncanonical allosteric AMP/ADP binding site is identified to stabilize the conformation of the regulatory flexible loop. Our findings not only explore a new mechanism of PRPS regulation with structural basis, but also propose an additional layer of cell metabolism through PRPS filamentation.