Polysaccharides are an important class of biological polymers as they provide structural support, store energy and mediate cell signaling. We are interested in understanding polysaccharide synthesis and transport across different domains of life. Specifically, we are interested in understanding bacterial cell wall and fungal cell wall synthesis. Peptidoglycan is an essential component of bacterial cell walls, while chitin and glucan are key components of fungal cell walls. These oligosaccharide/polysaccharide building blocks are synthesized intracellularly and then transported across the cell membrane for further assembly.
Lipid-linked oligosaccharide transport in bacterial cell wall synthesis and N-glycosylation
In bacteria, the synthesis and transport of lipid-linked peptidoglycan precursors involves multiple steps. Two essential steps are Lipid I production and Lipid II flipping, being carried out by integral membrane proteins MraY and MurJ, respectively. For over five decades, MraY has been considered a very promising target for the development of new antibiotics, as it is inhibited by five different classes of natural product antibiotics and a bacteriolytic phage. However, despite many years of effort, the development of antibiotics against MraY has stagnated, largely because of a poor structural and mechanistic understanding of MraY and the human MraY paralog GlcNAc-1-P transferase (GPT or DPAGT1). Furthermore, the mechanistic and structural basis of Lipid II flipping by MurJ has remained elusive. Our structural, biochemical, biophysical, and cell-based studies of these integral membrane proteins have shed light on their biochemical functions and provided insights into development of antibiotics with novel mechanisms of action.
Solving the Structures
Through crystallographic studies, the Lee Lab has solved the structure of apoenzyme MraY (Science 2013) and MraY bound to representative members of each class of its natural product inhibitors, including muraymycin (Nature 2016), capuramycin, caprazamycin, and mureidomycin (Nat Comm 2019). We also solved the structure of MraY paralog GPT in complex with tunicamycin (Nat Struc Mol Biol 2018). The Lee Laboratory determined the first structure of MurJ (Nat Struc Mol Biol 2016) and took crystallographic snapshots of MurJ at multiple stages of lipid flipping (Nat Comm 2019).
Our Discovery
Our structural and mechanistic studies have provided new insights into the mechanisms of MraY inhibition by natural product antibacterials. We have developed a comprehensive model of the key hotspots for MraY inhibition that can be employed to rationally design high-affinity inhibitors of MraY with favorable physicochemical properties. We anticipate that our work will propel forward the development of antibiotics against this important and validated target.
GPT catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and is the target of the natural product tunicamycin. Tunicamycin shows potent antibacterial activity by inhibiting MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We have demonstrated that this difference could be exploited to design inhibitors as potential antibiotics by developing a tunicamycin derivative selective for MraY (J. Mol Biol 2020).
The molecular identity of the Lipid II flippase was only recently revealed and the structural basis of Lipid II flipping by MurJ was previously unclear. Our structural analyses and mutagenesis studies have provided the basis of Lipid II recognition by MurJ and are the first to show that an alternate access mechanism is utilized for Lipid II flipping. Furthermore, we have captured the structures of MurJ in multiple states of its transport cycle, which has allowed us to visualize the conformational transitions this flippase undergoes while transporting Lipid II across the bacterial cell wall. (Annu. Rev. Biochem. 2022) (Nat. Comms. 2019)
Chitin production and transport in fungal cell wall synthesis
In fungal cell wall synthesis, chitin is synthesized by integral membrane chitin synthases (Chs’s). Chs’s catalyze both the homopolymerization of GlcNAc, using UDP-GlcNAc as a substrate, and the extrusion of nascent chitin polymer across the membrane to the extracellular side. Because chitin and Chs are not present in vertebrates and plants, Chs has been pursued as the target for antifungal agents for clinical and agricultural use. However, the mechanisms of chitin synthesis and inhibition by Chs-targeted antifungal agents such as Nikkomycin Z and polyoxin D, remain unclear due to the lack of Chs structures. In collaboration with Ken Yokoyama’s lab in Duke Biochemistry, we provide the structural basis of Chs regulation and inhibition by antifungal agents nikkomycin Z and polyoxin D and offer an unexplored avenue for the development of antifungal drugs.
Solving the Structures
Through cryo-EM studies, the Lee lab has solved the first structure of Chs2 in apo and substrate-bound states as well as bound to two representative antifungal inhibitors, Nikkomycin Z and polyoxin D (Nat Struc Mol Biol 2022).
Our Discovery
In collaboration with Ken Yokoyama’s lab in Duke Biochemistry, we have unveiled the basis of substrate and inhibitor recognition and provided insights into the chemical logic of Chs inhibition by Chs-targeted antifungal drugs. In addition to discovering an unexpected mechanism of lipid-mediated Chs regulation, we have also identified new hotspots of Chs that can be rationally targeted for development of novel Chs inhibitors.