Structure-based mechanism of lipoteichoic acid synthesis by Staphylococcus aureus LtaS

Abstract

Staphylococcus aureus synthesizes polyglycerol-phosphate lipoteichoic acid (LTA) from phosphatidylglycerol. LtaS, a predicted membrane protein with 5 N-terminal transmembrane helices followed by a large extracellular part (eLtaS), is required for staphylococcal growth and LTA synthesis. Here, we report the first crystal structure of the eLtaS domain at 1.2-Å resolution and show that it assumes a sulfatase-like fold with an ␣/␤ core and a C-terminal part composed of 4 anti-parallel ␤-strands and a long ␣-helix. Overlaying eLtaS with sulfatase structures identified active site residues, which were confirmed by alanine substitution mutagenesis and in vivo enzyme function assays. The cocrystal structure with glycerolphosphate and the coordination of a Mn 2؉ cation allowed us to propose a reaction mechanism, whereby the active site threonine of LtaS functions as nucleophile for phosphatidylglycerol hydrolysis and formation of a covalent threonine-glycerolphosphate intermediate. These results will aid in the development of LtaSspecific inhibitors for S. aureus and many other Gram-positive pathogens.

Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria

“…The 3D structure of the extracellular catalytic domain of LtaS has been determined (17,20). Overall, extracellular catalytic domain of LtaS (eLtaS) assumes a sulfatase-like fold; however, its active site is distinct from that of sulfatases (20).…”

“…The 3D structure of the extracellular catalytic domain of LtaS has been determined (17,20). Overall, extracellular catalytic domain of LtaS (eLtaS) assumes a sulfatase-like fold; however, its active site is distinct from that of sulfatases (20). Threonine (T300) of LtaS together with residues E255, D475, and H476 coordinate a manganese ion and assemble to form a binding pocket for glycerolphosphate (20).…”

“…Overall, extracellular catalytic domain of LtaS (eLtaS) assumes a sulfatase-like fold; however, its active site is distinct from that of sulfatases (20). Threonine (T300) of LtaS together with residues E255, D475, and H476 coordinate a manganese ion and assemble to form a binding pocket for glycerolphosphate (20). As revealed from the cocrystal structure of eLtaS with glycerol-phosphate, one oxygen atom of the phosphate group is coordinated with Mg 2+ , whereas the remainder of the phosphate group is stabilized by hydrogen bonding with H416 and W354.…”

Systematic identification of conserved bacterial c-di-AMP receptor proteins

“…Cultures were grown to an OD 600 of 0.5-0.7, protein expression induced with 0.5 mM IPTG, and incubated overnight at 16°C. Protein purifications were performed by nickel affinity and size exclusion chromatography as previously described (11,39). Protein concentrations were determined by A 280 readings.…”

Profiling and tandem mass spectrometry analysis of aminoacylated phospholipids in Bacillus subtilis

“…First, the D-alanylated lipid may be produced by DltD, and transported to the outer leaflet by a flippase such as the integral membrane protein DltB or the pore domain of MprF which is known to transport L-lysyl-PG and other L-aminoacyl-PGs 9 . Second, D-alanyl-PG can be transferred to lipo- and wall-teichoic acids by a transferase such as DltB, a putative membrane-bound O-acyltransferase 39 , or incorporated as D-alanyl-glycerolphosphate units from D-alanyl-PG into the growing ends of teichoic acids by their respective polymerases LtaS 40, 41 and TagF 42, 43 . It is worth noting that D-alanyl-CL has been reported before 27 .…”