*EASTBIO* Probing novel aspects of mycolic acid transport and synthesis in Tuberculosis

BBSRC Theme: Bioscience for Health

Tuberculosis is one of the main causes of death worldwide, mainly due to the extensive and increasing antimicrobial resistance of Mycobacterium tuberculosis (Mtb). Therefore, there is an urgent need to elucidate essential biological processes and novel pathways, which are responsible for the survival and pathogenicity of tuberculosis.

The unusual mycobacterial cell wall lipids play a major role in pathogenesis. In particular, mycolic acids are unique lipid components of the cell envelope of all members of the Mycobacterium genus. Thus, proteins involved in their formation and transport are key virulence factors. MmpL and MmpS proteins mediate transport of these unique lipids across the mycobacterial inner-membrane and are also implicated to be involved in heme uptake, drug efflux and siderophore export1.

In this context deep understanding of the biosynthetic enzymes and subsequent transport of these unusual fatty acid features would pave the way for the elucidation of the detailed molecular mechanisms involved in the complex cell wall assembly. An early example could be seen for SQ109, a 1,2-diamine, molecule, which disrupts the mycobacterium cell wall assembly, and interferes with mycolic acids already residing within the cell wall2. Subsequent whole genome sequencing showed that all drug resistant mutants had mutations in the MmpL gene, the mycolic acid transmembrane transporter2. MmpL has no sequence conservation with any other known membrane protein, thus represents a novel protein family with not known 3D architecture homologs. Therefore, solution of the 3-dimensional x-ray structure of this mycolic acid transporter, would pave the way for the molecular-detailed design of novel inhibitors and most importantly would provide invaluable insights of the fundamental underlying mechanisms for the formation of the complex mycobacteria cell wall. In addition, studies on the mycolic acid biosynthetic machinery, a biological process, for which very little is known, will also highlight novel biochemical aspects. Structural and various biochemical studies will allow us to understand its substrate range, mechanism and possible inhibition.

This project lies right at the interface of biology, chemistry and physics with potential applications in bio-nanotechnology and medical sciences will focus on the biosynthesis pathway of mycolic acid and the structure and function of membrane proteins involved in the mycolic acid transport, investigating substrate specificity and potential inhibition. A multidisciplinary approach will be engaged and extensive training will be provided in advanced structural, biophysical and biochemical methods, spanning from x-ray crystallography and pulsed-EPR to mass spectrometry and single molecule methodologies to investigate gating of these important and challenging systems.

X-ray (hanging-drop) and lipidic cubic phase (LCP) crystallography will be used to obtain structural information to molecular detail and solve membrane protein structures in distinct conformational states, in order to unravel gating and elucidate function.
Pulsed electron paramagnetic resonance (EPR) spectroscopy will be also facilitated, also known as PELDOR or DEER to directly measure spin-to-spin distances at distinct conformational states, within these protein complexes and identify experimentally, their unknown oligomeric and conformational state, under a variety of conditions and molecular triggers. This method will be used in combination with site-directed spin labeling (SDSL), in which, single cysteine residues are engineered back to sites of interest and are subsequently modified with spin labels. The combination of these two advanced techniques is anticipated to provide high quality structural information, as previously demonstrated for the membrane protein channel MscS3.

Finally, sophisticated biochemical assays in combination with advanced mass spectrometry techniques3 will be used to identify potential substrates and inhibitors allowing characterisation of structure activity relationships and potentially their interaction with the protein. This information will feed back into obtaining a better understanding of the effect on protein function, which can also be monitored by single molecule methodologies3, allowing direct protein structure and function relationships to be created.

Important findings from this basic research project could potentially constitute a novel avenue for the fundamental understanding of key mechanisms of the enzymes in mycolic acid biosynthesis and its novel transporter.