Structural & biophysical characterisation of M. tuberculosis MmpL3

Tuberculosis (TB) is the leading cause of death from a single infectious agent, with a staggering 10 million people estimated to have contracted the disease in 2019 alone. Whilst treatments for TB exist, continual emergence of drug-resistant strains necessitates urgent development of next-generation antituberculars. A promising new target is the essential glycolipid exporter mycobacterial membrane protein large 3 (MmpL3), a member of the resistance-nodulation-division (RND) superfamily. MmpL3 is required for construction of the distinctive Mycobacterium tuberculosis (Mtb) cell envelope; a complex lipid-rich assembly critical to mycobac- terial viability. Despite discovery of numerous MmpL3 inhibitors, including one in clinical trials, a structural understanding of the transporter, and by extension the variants identified to confer resistance to said experimental agents, has remained elusive. To this end, the research herein presents a 3.0 Å reconstruction of Mtb MmpL3; the culmination of a project structurally pursuing the RND exporter through construct design & characterisation, in vitro binder selection, nanodisc reconstitution, in meso & in surfo crystallisation trials, and imaging by Cryo-EM. Unexpectedly ... (continues)