Membrane Protein Complexes in the M. Tuberculosis Divisome: Structures and Interactions

PROJECT SUMMARY Tuberculosis (TB) has inflicted a quarter of the worldwide population and caused 1.6 million deaths in 2021 alone. However, there have been no new classes of TB drugs developed since the 1970s. New therapies are desperately needed as multi-drug and extensive drug-resistant TB cases have increased rapidly. The primary reason for drug resistance is latency, where the causative agent, Mycobacterium tuberculosis (Mtb), remains non-proliferative in the patient’s body. Understanding the cell division process in Mtb through structural characterizations of the participating protein complexes is critical for designing resistance-breaking therapeutics and is the objective of this project. Bacterial cell division is mediated by the divisome, which comprises dozens of proteins spanning the cytoplasmic membrane and the periplasm, with the Z-ring as the scaffold. We recently discovered that the cytoplasmic N-terminal region of Mtb FtsQ binds FtsZ, which is the protomer of the Z-ring, and hypothesized that FtsQ may stabilize the curvature of the Z-ring and anchor the Z-ring to the inner membrane. In E. coli, FtsQ forms a ternary complex with FtsB and FtsL to activate enzymes that are responsible for the synthesis of cell walls. However, the amino-acid sequences of the Mtb FtsQ, FtsB, and FtsL are distinct from their E. coli analogs, suggesting different structures and functional mechanisms. The Specific Aims of this project are: (1) to investigate the structural and functional roles of the FtsQ-FtsZ interaction; (2) to characterize the structure, interaction, and function of the FtsQ-FtsB-FtsL complex; and (3) to develop 17O NMR for probing membrane protein interactions. This work will employ advanced solid-state and solution NMR combined with molecular dynamics simulations to characterize protein complexes in native-like membrane environments. The proposed functional studies will establish crucial connections between structural insights and biological activities, with profound implications for drug resistance. Our interdisciplinary team, with expertise in NMR spectroscopy, computational biophysics, and microbiology, is ideally positioned to accomplish the proposed studies. This research will yield critical knowledge on the cell division process in Mtb and help identify drug targets for effective therapeutic strategies to address the TB epidemic. The new methodologies that we will develop represent the frontier of protein structural biology. 1