Structural characterization of three novel proteins from Mycobacterium species vital for their survival

Abstract

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), is one of the deadliest human pathogens, with one third of the world’s population presently infected, resulting in 1.3 million deaths in 2012. The emergence of multiple drug resistant strains of M. tuberculosis has resulted in an inability to control and treat this pathogen. Accordingly, there is an urgent need for the development of new anti- TB therapeutics. Structural genomics has played a pivotal role in the characterization of the M. tuberculosis proteome to gain functional insight into pathogenesis, with applications for future rational drug design. While TB structural genomics has generated data on a number of mycobacterial targets, structural data is missing for up to 90% of the M. tuberculosis proteome. To gain a global understanding of mycobacterial pathogenesis, an in-depth structural characterization of the M. tuberculosis proteome is required. My work described here explores the structural characteristics of three novel proteins essential for mycobacterial growth and host survival. Utilizing applications of x-ray crystallography, crystal structures of each target were determined, and have been utilized to characterise drug inhibition mechanisms, as well as provide functional insight into the biological role of the targets. The key first line anti-TB drug, isoniazid, exhibits inhibition against multiple M. tuberculosis targets, where resistance is readily developed. Rv2971, an essential aldo-keto reductase, is a recently identified target of isoniazid. To gain structural insight into isoniazid inhibition mechanisms, the crystal structure of Rv2971 was determined to 1.6 A, revealing the structural architecture of the isoniazid and NADPH binding sites, paving the way for future structural characterization of inhibition. A recently characterized drug, tetrahydrolipstatin (THL), has been found to strongly inhibit Rv3802c, an essential lipase involved in mycolic acid biosynthesis. To characterise mechanisms of THL inhibition, the crystal structure of Rv3802c in complex with THL was determined to 2.9 A. The crystal structure reveals the binding mechanisms of THL against an essential mycobacterial protein. Identification and structural characterization of addition THL drug targets will allow for future THL based drug design. A high percentage of the M. tuberculosis proteome is annotated as “hypothetical” proteins. To truly understand the mechanism of mycobacterial pathogenesis, further characterisation of hypothetical proteins is required. Rv0807, and its M. smegmatis orthologue, MSMEG_5817, have recently been identified as essential for mycobacterial survival within macrophages. The gene is annotated as a hypothetical protein, with little known on its functionality. The crystal structure of MSMEG_5817 was, via Se-MAD approach, successfully determined to 1.7A. The structure shares similarities with sterol carrier proteins (SCP), which bind and transport biologically relevant lipids. The lipid binding capabilities of MSMEG_5817 was probed, revealing binding to a number of phospholipids in a differing binding mechanism to the SCPs. The crystal structure determined structurally represents this new class of mycobacterial proteins, providing further insight into mechanisms of host survival. The three crystal structures provide insight into the drug binding mechanisms and functional characteristics of each target, allowing for further characterization of the novel mechanisms of mycobacterial pathogenesis, with potential for development of new anti-TB therapeutics.