Molecular mechanism of MFS and MOP lipid transporters in cell wall biosynthesis. - PROJECT SUMMARY/ABSTRACT Transporters from the multidrug exporter/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily and the major facilitator superfamily (MFS) are central to multiple cell wall biopolymer biosynthesis pathways. Transporters from both superfamilies exhibit significant differences in their architecture, lipid substrate selectivity, and transport mechanisms. There are several critical gaps in our current understanding of the molecular mechanism of substrate recognition and transport, substrate specificity, ion-coupling, and activity modulation of lipid transporters from these two superfamilies. This project seeks to investigate the structure and function of two lipid transporters involved in teichoic acid synthesis: the glycolipid transporter LtaA from the MFS superfamily and the teichoic acid transporter TacF from the MOP superfamily. Thus far, my work has revealed the structure of LtaA in an outward-facing apo state. Additionally, computational methods, cysteine disulfide trapping, and AlphaFold modelling have been employed to validate inward-facing models of LtaA. Our findings revealed that in both outward- and inward-facing conformations, this protein displays an amphipathic central cavity crucial for diglucosyl-diacylglycerol transport, and suggest that LtaA employs a ‘trap-and-flip’ mechanism to facilitate glycolipid translocation. In contrast, there is currently a lack of structural and functional data regarding the mechanism of the MOP teichoic acid transporter TacF, and there are still many questions unanswered about the molecular mechanism of lipid transporters from both superfamilies. We have additionally explored the ion- coupled lipid transport mechanism of LtaA, recognizing its essential role in modulating the cell wall composition under acidic conditions. We hypothesize that ion-coupled co-transport of lipids may be linked to adapting the cell wall composition. However, there are few detailed studies on the precise mechanism of ion-coupling for MFS and MOP lipid transporters. In this application we want to reveal how LtaA and TacF select for their lipid substrate at the atomic level, elucidate their ion coupling mechanisms, and reveal the impact of membrane lipids on transport. Collectively, our proposed research will broadly impact the field of lipid transport by characterizing the molecular mechanism of transporters from two superfamilies commonly associated to cell wall biopolymer biosynthesis pathways. These studies have the potential to uncover novel molecular mechanisms underlying lipid and ion co-transport, which will be critical for understanding the function of cell wall lipid transporters in bacteria, and will potentially accelerate the structure-based drug design of activity modulators targeting these proteins.
