Structural & biophysical characterisation of M. tuberculosis MmpL3

Thesis

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 monomeric, the Mtb MmpL3 atomic model provides insights into the evolutionary co-option of RND architecture for lipophile transit. Most notably, the protein houses an extensive periplasmic cavity with putative functional rel- evance as the glycolipid translocation conduit. A detergent molecule resolved within the cavity plausibly sheds light on interactions employed by MmpL3 to coordinate its native substrate. Additionally, comprehensive mapping of known resistance-conferring substitutions suggests the existence of at least two routes, operant within different domains, by which antituberculars inhibiting MmpL3 can be mutationally circumvented. Taken together, the data reported in this thesis set the stage for an era of structure-based drug design against MmpL3; guided by an enhanced appreciation of the underlying resistance landscape.

Authors: Adams, O

• DOI: 10.5287/ora-ozgron2qy
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Tuberculosis; Sybodies; Antitubercular; Membrane Protein; Drug Resistance; MmpL3; MmpL; Nanodiscs; Mycolic Acid; LMNG; Cryo-EM; Nanobodies; RND Transporter
• Subjects: Biochemistry; Structural Biology