Tuberculosis kills 1.5-2 million people globally every year. An effective vaccine or chemotherapy has yet to be developed. Recently, through a large-scale transposon mutagenesis screening, the Mycobacterium tuberculosis (Mtb) proteasome and the proteasomal ATPase (Mpa) were found to be required for Mtb resistance to killing by a source of nitric oxide (NO). NO is produced by the host immune system to control Mtb infections. Proteasome and Mpa appear to protect Mtb against NO by degrading proteins after exposure to NO. Thus, Mpa and the Mtb proteasome may be promising targets for the development of anti-Tb chemotherapeutics. Mtb proteasome has unique broad substrate specificity towards small synthetic peptides, and its proteolytic activity is inhibited by MLN-273, an analog of the anti-myeloma drug bortezomib that targets human proteasome. The unique substrate binding property of Mtb proteasome can be exploited to develop Mtb specific inhibitors. Mpa is homologous to ATPases that function with the eukaryotic proteasome, and likely unfolds the protein substrate for processive degradation by Mtb proteasome. A mutant Mpa protein missing only its last two amino acids retains ATPase activity, yet fails to protect Mtb against nitrite. We propose to use a combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography to investigate the structures of Mtb proteasome and Mpa. Our comprehensive structural studies (1) will reveal the structural basis of the Mtb proteasome's broad substrate specificity; (2) will uncover the inhibition mechanism of the dipeptidyl boronate MLN-273; (3) will elucidate the conformational changes of Mpa associated with the ATP hydrolysis cycle; (4) will provide answer to the long standing question as to how a symmetry mismatched six-fold ATPase oligomer activates the seven-fold proteasome, (5) will contribute to a better understanding of the eubacterial proteasome biology, and (6) will set the stage for the structure-based anti-TB chemotherapeutic development. ? ? ?
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