The Structural Biology of Lipid Transport in Cell Wall Synthesis and N-glycosylation
Both Lipid I production and Lipid II flipping are essential steps in bacterial cell wall synthesis. These two processes are 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 MraY-target antibiotics has been stagnant largely because of a lack of a structural and mechanistic understanding of MraY function, inhibition, and similarity to human MraY paralog human 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 provide insights into developing 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. Communs. 2019). We also solved the structure of MraY paralog GPT in complex with tunicamycin (Nat Struc Mol Biol 2018). The Lee Lab determined the first structure of MurJ (Nat. Struc. Mol. Biol. 2016) and took crystallographic snapshots of MurJ at multiple stages of lipid flipping (Nat. Communs. 2019).
Our structural and mechanistic studies have provided new insights into the mechanisms of MraY inhibition by antibacterial natural products. We have developed a comprehensive model of the key hotspots of 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 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.
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.