Investigation of the mechanistic basis of substrate recognition and translocation by the MFS flippase LtaA

Lipoteichoic acids (LTA) are essential cell-wall components in Gram-positive bacteria, including the human pathogen Staphylococcus aureus. They contribute to cell growth, cell stability, virulence and cell division. Interrupting their biogenesis pathway leads to severe growth defect such as longer LTAs, larger cells, and misplaced septa. S. aureus LTA is composed out of a polyglycerol chain on a gentiobiosyl-diacylglycerol anchor. Their synthesis starts on the cytoplasmic leaflet of the membrane by the glycosyltransferase YpfP, followed by translocation of the lipid-linked disaccharide across the plasma membrane, followed by polymerization of the chain at the extracellular side of the membrane. Genetic evidence has suggested that LtaA is the flippase responsible for the translocation of the lipid-linked disaccharide. In the first part of this thesis, we confirm that LtaA is indeed the flippase performing the translocation of gentiobiosyl-diacylglycerol anchor during LTA biosynthesis. We present the outward-open crystal structure of LtaA and identify that LtaA contains the Major Facilitator Superfamily (MFS) topology. Investigation of the structure showed that LtaA contains a amphiphilic cavity, which was never observed before. In this cavity the N-terminal cavity consist of polar residues whereas the C-terminal cavity is composed of hydrophilic residues. Using our established in vitro flipping activity assay, and S. aureus ∆ltaA strains, we demonstrate the importance of the hydrophobic cavity. We show that LtaA is a proton-coupled antiporter, which is essential for S. aureus to combat physiological acidic stress conditions. Elucidation of the LtaA structure identified the first MFS flippase, and the first proton-coupled flippase. Flipping of lipids with large polar headgroups is an energetically costly reaction, that is frequently coupled to ABC transporters. However, ion-coupled lipid transporters of the MFS, such as LtaA, have been shown to play essential roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Structures of MFS transporters showed overlapping architectures pointing to a common mechanism. Whereas studies on ATP-driven transporters have shown to operate via a ‘trap-and-flip’ or ‘credit-card’ mechanism, the mechanism of ion-coupled MFS lipid transporters is largely unknown. The second part of this thesis investigates the flipping mechanism used by LtaA as a representative system of MFS flippases. We show using cysteine-crosslinking that LtaA adopts alternating conformations, and that alternating access to the cavity is essential for transport. We reveal that LtaA adopts asymmetric openings with distinct functional relevance during transport of the glycolipid. We characterized the hydrophobic cavity using in vivo and in vitro assays, showing that the entire amphipathic cavity of LtaA contributes to lipid binding. Although, the N-terminal hydrophilic pocket imposes the substrate specificity. Based on these results, we propose that LtaA catalyzes lipid translocation using a ‘trap-and-flip’ mechanism that might be shared within the MFS lipid transporters subfamily. Taken together, a thorough investigation of LtaA is performed in this thesis. Our results unveil important structural and mechanistic details showing that LtaA recognizes the gentiobiosyl-diacylglycerol by its gentiobiosyl headgroup and adapts an asymmetric lateral opening to load the substrate into the cavity. In the cavity the sugar headgroup interacts with the N-terminal hydrophilic pocket, and the lipid tails are embedded in the hydrophobic C-terminal pocket. After conformational changes, the substrate is released via both lateral gates at the extracellular side. This is followed by protonation of LtaA, inducing conformational changes to an inward-facing conformation. These mechanistic results provide foundations for developments of new strategies to counteract live-threating S. aureus infections, as well as a basis for drugs and lipid-linked-bioactive molecules targeting cells expressing pharmacologically important proteins from the MFS