Structural Studies of Bacterial Cell Wall Synthesis and Remodeling
Abstract
Bacterial peptidoglycan (PG) cell wall is an essential structural element, providing structural integrity and protection. The essentiality and uniqueness of PG make it an ideal target for antibiotics as over 50% of all antibiotics in clinical use target the PG biosynthesis pathway. PG synthesis involves precursor synthesis in the cytoplasm, transport across the membrane, and final assembly in the periplasmic space to form functional peptidoglycan. PG is also a dynamic structure, as it requires synthesis and degradation in a highly coordinated manner to prevent cell lysis. Despite decades of research, the cellular mechanisms regulating peptidoglycan synthesis and remodeling machineries have remained largely mysterious, particularly at the membrane-bound steps.To understand this essential process, we propose to investigate the structure, mechanism, and inhibition of the proteins and protein complexes involved in the PG pathway during cell division. My dissertation study focuses on key enzymes involved in PG precursor translocation (MraY), transport (MurJ), assembly (FtsBLQWI) and remodeling (FtsEX-EnvC). We employed single-particle cryo-electron microscopy (cryo- EM) to study the inhibition of MraY and MurJ, and to elucidate the regulatory mechanisms of protein complexes FtsBLQWI and FtsEX-EnvC from structural biology standpoint. We utilized in vitro biochemical assays to characterize the biochemical function of these protein complexes. The first part of the work studies PG precursor translocase and transport proteins MraY and MurJ, respectively. MraY catalyzes the reaction that anchors precursor on the membrane, while MurJ then flips the precursor across membrane to the periplasm. MraY has been a target for many naturally occurring nucleoside inhibitors. Rational design and synthesis of natural inhibitor analogs have been a strategy for discovering new and better antibiotics. In collaboration with Dr. Satoshi Ichikawa, we designed and constructed libraries of natural MraY inhibitor analogs. We identified promising MraY inhibitors from these libraries and several showed potent inhibitory and antibacterial activity. We reported two high-resolution cryo-EM structures of MraY bound to the new inhibitor analogs. The structures reveal a novel mode of inhibition, which provides a promising direction in antibiotics design. MurJ has been reported to be inhibited by chemically diverse compounds, but the mechanisms of MraY inhibition by these compounds have been elusive. Understanding MurJ inhibition is important because MurJ is considered a promising new drug target for antibiotic development. We attempted to elucidate the inhibition mechanism of MurJ by capturing MurJ with inhibitor(s) bound using X-ray crystallography. However, despite numerous attempts, the structural characterization of MurJ with inhibitors remained challenging. To overcome the challenges, we generated initial cryo-EM data and proposed strategies for future structural studies of MurJ using cryo-EM. The second part explores the core PG synthesis enzyme complex FtsWI and the accessory regulatory complex FtsBLQ, aiming to understand the interaction and regulation of these essential five proteins during cell division. The molecular regulation within the complex is unknown due to lack of structural information. We attempted to obtain the structure of FtsBLQWI using cryo-EM to elucidate the regulatory mechanism. I co-expressed and obtained homogeneous protein complex from an overexpression system. I developed a functional assay that circumvented the problem of substrate inaccessibility by in situ synthesis of the substrate and demonstrated that the purified complex had robust activity. While still in progress, we identified biochemical condition that yielded promising results for 3D reconstruction, laying the groundwork for future structural investigation on FtsBLQWI. The final part delves into the mechanism of FtsEX-EnvC complex function, which regulates the degradation of PG during cell division. FtsEX-EnvC complex controls PG remodeling by regulating the enzymes that hydrolyze PG. However, the molecular details of how this complex work is unknown. We obtained the cryo-EM structure of the full-length FtsEX-EnvC complex in different nucleotide states, revealing an unexpected conformation in the presence of ADP and providing a novel model on the activation mechanism through spatial regulation. Overall, the study provides structural, inhibitory, and mechanistic insights into several components of the PG pathway. Peptidoglycan synthesis and remodeling are not only fundamental processes in bacterial biology, but also a rich source of antibiotic drug targets. In light of rapidly emerging antimicrobial resistance, understanding the pathways governing PG synthesis and remodeling is crucial for discovering novel targets to develop new inhibitors.
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Hao, Aili (2023). Structural Studies of Bacterial Cell Wall Synthesis and Remodeling. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/29161.
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