Understanding the structural and functional implications of lysine succinylation in Mycobacterium tuberculosis heat shock protein 16.3.

Heat shock protein 16.3 (Hsp16.3), a major immunodominant antigen of Mycobacterium tuberculosis, exhibits molecular chaperone function that is essential for pathogen's survival and slow growth inside hosts, as well as for enhancing the efficacy of Bacillus Calmette-Guérin (BCG) vaccine. Proteomic studies revealed that Hsp16.3 undergoes lysine succinylation in vivo at all lysine residues (K47, K64, K78, K85, K114, K119 and K132) except K136. However, the effects of succinylation on its structure and function remain unexplored. This study investigated the impact of succinylation, induced by physiological (succinyl-CoA) and/or non-physiological (succinic anhydride) donors, on the structure, stability and chaperone function of Hsp16.3. Succinylation of all eight lysine residues, affirmed via fluorescamine assay and mass spectrometry, led to structural (secondary and tertiary) alterations, as indicated by circular dichroism (CD), fluorescence and in-silico analyses. Succinylation induced oligomeric dissociation (dodecamer to dimer) and enhanced surface hydrophobicity of Hsp16.3. Moreover, succinylation reduced protein stability, making it more conformationally flexible and less compact, as revealed by urea-denaturation, chymotrypsin-digestion and computational studies. Despite this reduced stability, succinylated Hsp16.3 exhibited enhanced chaperone activity, offering improved protection to stressed-prone client proteins. These findings provide useful insights into this modification, offering potential therapeutic avenues for targeting Hsp16.3 in M. tuberculosis infection.