Abstract
Tuberculosis is the most lethal infectious disease globally, despite having successful treatment options. Understanding the mechanism TB undergoes to evade the defenses of its human host is vital for new drug discovery and reducing the number of cases of drug-resistant TB. Rv2224c is a Mycobacterium tuberculosis protein identified as being necessary for the pathogen to survive inside macrophages. Here we showed that refolded purified Rv2224c has protease activity and identified its direct substrate in vitro is heat-shock protein GroEL2, which is predicted to be a highly antigenic molecular chaperone and also a candidate substrate in vivo. To further explore the enzymatic properties of Rv2224c, the inactive mutant Rv2224c S228A was bound to GroEL2 and cocrystallized for structural characterization. However several detrimental issues were analyzed such as the destabilizing and inhibitory effect of co-solvent isopropanol and a mutation in the cpn60.2 gene encoding GroEL2. Sparse matrix screens would be necessary to find different conditions to crystallize Rv2224c in, including with its substrate, GroEL2 or inhibitor, AEBSF.