Structural and mechanistic investigation of a proton-dependent lipid transporter involved in lipoteichoic acids biosynthesis

Staphylococcus aureus (S.aureus) is a successful opportunistic human pathogen, causing superficial infections such as skin and soft tissue infections as well as invasive fatal ones including endocarditis, pneumonia and septicemia with high mortality. It represents one of the growing public health concerns worldwide since the rapid spreading of multi-antibiotic resistant S.aureus strains has increased the failure of therapeutics, urgently calling for pathogenesis research and development of effective treatment strategies.
30% of the population carries S.aureus on the skin and mucous membranes where the pH is usually mild acidic (average 4 to 6). Managing survival under this environment is a critical step for S.aureus successful colonization, dissemination and infection. The cell wall, a multi-layered protective structure, plays a crucial role in maintaining S.aureus viability under hostile surroundings. Lipoteichoic acids (LTA) are one type of the main components of the S.aureus cell wall, composed of repeating glycerol phosphate units and a glycolipid anchor called diglucosyl-diacylglycerol (Glc2-DAG) that fixes LTA polymers on the outer leaflet of the plasma membrane. Glc2-DAG is synthesized in the cytoplasm and transferred across the plasma membrane by the integral membrane protein LtaA (lipoteichoic acid protein A). The deletion of LtaA in S.aureus led to the alteration of the LTA anchor from Glc2-DAG to diacylglycerol (DAG) and attenuated virulence during animal infection.
LtaA belongs to transporters that mediate glycolipid translocation. This category is closely involved in the cell wall biosynthesis by transferring multiple precursors or molecules to satisfy the proper assembly of the cell wall. Bacteria harbor diverse glycolipid transporters, most of which are not well understood. LtaA was predicted to belong to the major facilitator superfamily (MFS), a large family of membrane proteins that are ubiquitously distributed in all kingdoms of life, transferring a broad range of substrates from sugars, peptides to ions and lipids across membranes. Research on MFS transporters involved in glycolipids transport is scarce, hindering the understanding of their flipping mechanisms.
In this study, we determined the structure of LtaA by X-ray crystallography at a resolution of 3.3 Å. LtaA presents the canonical MFS fold with 12 transmembrane helices (TMs) arranged in two pseudo-symmetric sub-domains, N-domain (TM1-6) and C-domain (TM7-12). A striking feature is the presence of a large amphiphilic central cavity which we hypothesized to accommodate the amphiphilic substrate Glc2-DAG. By analyzing LtaA crystal structure, along with site-direct mutagenesis and transport assays in vitro, we demonstrated that the di-glucosyl moiety of Glc2-DAG is recognized by multiple conserved hydrophilic residues located in the N-terminal domain and loaded to the central cavity. We also proposed a proton-coupling mechanism where E32 undergoes protonation/deprotonation, and this extra driving force allows Glc2-DAG to be translocated at a higher rate. By investigating LtaA function in S.aureus, we revealed that the proton-coupling mechanism allows LtaA to act as an environmental pH sensor and contribute to the survival of S.aureus under an acidic environment. Our results provided insights into the molecular basis of Glc2-DAG flipping and made LtaA a novel target for the development of anti-S.aureus therapeutics.