Abstract
The divergence of archaea, bacteria and eukaryotes was a fundamental step in evolution. One marker of this event is a major difference in membrane lipid chemistry between these kingdoms. Whereas the membranes of bacteria and eukaryotes primarily consist of straight fatty acids ester-bonded to glycerol-3-phosphate, archaeal phospholipids consist of isoprenoid chains ether-bonded to glycerol-1-phosphate. Notably, the mechanisms underlying the biosynthesis of these lipids remain elusive. Here, we report the structure of the CDP-archaeol synthase (CarS) of Aeropyrum pernix (ApCarS) in the CTP- and Mg2+-bound state at a resolution of 2.4 Å. The enzyme comprises a transmembrane domain with five helices and cytoplasmic loops that together form a large charged cavity providing a binding site for CTP. Identification of the binding location of CTP and Mg2+ enabled modeling of the specific lipophilic substrate-binding site, which was supported by site-directed mutagenesis, substrate-binding affinity analyses, and enzyme assays. We propose that archaeol binds within two hydrophobic membrane-embedded grooves formed by the flexible transmembrane helix 5 (TM5), together with TM1 and TM4. Collectively, structural comparisons and analyses, combined with functional studies, not only elucidated the mechanism governing the biosynthesis of phospholipids with ether-bonded isoprenoid chains by CTP transferase, but also provided insights into the evolution of this enzyme superfamily from archaea to bacteria and eukaryotes.
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Acknowledgements
We thank the staff of the BL19U1 beamline at the National Center for Protein Sciences Shanghai (NCPSS) at the Shanghai Synchrotron Radiation Facility for assistance with data collection. We thank J Chai for critical comments. We thank Peter Fodran and Adriaan Minnaard for the synthesis of DGGGP. This work was funded by the National Natural Science Foundation of China (NSFC; 31570842), the National Young Thousand Talents Program of China, and the Sichuan Province Thousand Talents Program to WC. AC was supported by a research program from the biobased ecologically balanced sustainable industrial chemistry (BE-BASIC). Computational support was provided by the special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase; U1501501).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Phylogenetic tree distribution of members of the archaeal and bacterial CTP transferases families. (PDF 423 kb)
Supplementary information, Figure S2
Amino acid sequence alignment of CarS homologs in three life kingdoms. (PDF 1042 kb)
Supplementary information, Figure S3
Detection of products catalyzed by ApCarS using high performance liquid chromatography-mass spectrometry (HPLC-MS). (PDF 195 kb)
Supplementary information, Figure S4
Activity assays of ApCarS in the presence of different ions. (PDF 281 kb)
Supplementary information, Figure S5
Stabilization of cytoplasmic loop 1 (CL1) is essential for the CTP-binding and catalytic activity of ApCarS mutants. (PDF 396 kb)
Supplementary information, Figure S6
Electron density maps for the structure of ApCarS bound to CTP and metal ions. (PDF 564 kb)
Supplementary information, Figure S7
The stabilization of the periplasmic region is required for the enzymatic activity of ApCarS. (PDF 302 kb)
Supplementary information, Figure S8
Isothermal titration calorimetry (ITC) measurements of the CTP-binding activity of ApCarS encoding amino acid substitutions in the cytoplasmic domain (CPD). (PDF 848 kb)
Supplementary information, Figure S9
Structural basis for this catalytic mechanism. (PDF 900 kb)
Supplementary information, Figure S10
Structural analysis the potential binding site of Archaeol. (PDF 208 kb)
Supplementary information, Table S1
Data collect and refinement statistics (PDF 116 kb)
Supplementary information, Table S2
Representative affinity constants and binding parameters demonstrating the effect of amino acid substitutions within the cytoplasmic domain (CPD) of ApCarS on CTP-binding (PDF 70 kb)
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Ren, S., Caforio, A., Yang, Q. et al. Structural and mechanistic insights into the biosynthesis of CDP-archaeol in membranes. Cell Res 27, 1378–1391 (2017). https://doi.org/10.1038/cr.2017.122
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DOI: https://doi.org/10.1038/cr.2017.122
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