The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

Abstract

Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of phosphatidylglycerol biosynthesis. Mutations in pgsA frequently correlate with daptomycin resistance in Staphylococcus aureus and other prevalent infectious pathogens. Here we report the crystal structures of S. aureus PgsA ( Sa PgsA) captured at two distinct states of the catalytic process, with lipid substrate (cytidine diphosphate-diacylglycerol, CDP-DAG) or product (PGP) bound to the active site within a trifurcated amphipathic cavity. The hydrophilic head groups of CDP-DAG and PGP occupy two different pockets in the cavity, inducing local conformational changes. An elongated membrane-exposed surface groove accommodates the fatty acyl chains of CDP-DAG/PGP and opens a lateral portal for lipid entry/release. Remarkably, the daptomycin resistance-related mutations mostly cluster around the active site, causing reduction of enzymatic activity. Our results provide detailed mechanistic insights into the dynamic catalytic process of PgsA and structural frameworks beneficial for development of antimicrobial agents targeting PgsA from pathogenic bacteria.

Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase

“…The pellets were then solubilized by 1% (w/v) dode maltoside at 4 °C for 1 h with mild agitation. The mixture was heated at 65 °C for 30 m [42][43][44] using the ARCIMBOLDO_LITE algorithm [45] and molecular replacement with models constructed by evolutionary coupling [46,47] Here, we demonstrate that Se-urea is a compatible and effective heavy-atom compound for the experimental phasing of crystals grown in LCP. With a single crystal, the structure of PlsY was successfully determined by Se-SAD.…”

“…The majority of the structures are solved by molecular replacement (MR). The methods for the two 'others' are Ab initio[42][43][44] using the ARCIMBOLDO_LITE algorithm[45] and molecular replacement with models constructed by evolutionary coupling[46,47], as shown in (B). Brackets indicate the number of structures in each category.…”

Mechanistic Studies and In Vivo Efficacy of an Oxadiazole-Containing Antibiotic

“…PgsA is an essential protein in S. aureus because of its important role in phospholipid synthesis in the bacteria . Additionally, PgsA shares 29% sequence identity with the human cardiolipid synthase which is responsible for the conversion of cytosine diphosphate diacylglycerol (CDP-DAG) and PG to cardiolipin and cytosine monophosphate (CMP) in mitochondria …”

The catalytic and structural basis of archaeal glycerophospholipid biosynthesis

“…The structure of SaPgsA appears highly reminiscent of the MtPis crystal structure described above (Grāve et al 2019;Yang et al 2021). The phosphatidyl moiety of the co-crystallized PGP and CDP-DAG moieties is positioned in virtually the same way.…”

“…E. coli Pgs (PgsA) contains the hallmark CAPT-motif and is predicted to form 6 transmembrane helices forming an integral membrane protein which is typical for members of the CAPT family (Chang and Kennedy 1967 ; Hirabayashi et al 1976 ). Only recently, the first crystal structure of a bacterial PgsA has been reported for Staphylococcus aureus (SaPgsA) (Yang et al 2021 ). PI synthesis has been studied in more detail and three structures of bacterial Pis are deposited in PDB (Clarke et al 2015 ; Grāve et al 2019 ; Belcher Dufrisne et al 2020 ) (Fig.…”