The C‐terminal extension of Mycobacterium tuberculosis Hsp16.3 regulates its oligomerization, subunit exchange dynamics and chaperone function

Abstract

Mycobacterium tuberculosis is a human pathogen that secretes a major immunodominant antigen, namely Hsp16.3, throughout the course of infection. Hsp16.3 belongs to the small heat shock protein family and exhibits a molecular chaperone function that is important for the growth and survival of M. tuberculosis in host cell macrophages. The importance of the N-terminal region for the structure and chaperone function of Hsp16.3 is well understood. However, the effect of the C-terminal region on these properties is far from clear. Therefore, we cloned, over-expressed and purified wild-type and seven C-terminal-truncated mutant proteins of Hsp16.3. Mutants with deletions of one and two C-terminal extension (CTE) residues had a structure and chaperone function similar to wild-type protein. Intriguingly, deletion of three residues from the CTE triggered perturbation of the tertiary structure, dissociation of the oligomeric assembly (dodecamer to octamer and dimer), enhancement of subunit exchange dynamics and improvement in the chaperone function of Hsp16.3. Interestingly, these structural modulations (except oligomeric dissociation) as well as chaperoning strength reached their apex upon truncation of the entire CTE ( RSTN ). Further deletions from the C-terminal region beyond the CTE increased only the degree of oligomeric dissociation, and the complete removal of this region made the protein into a dimer. Overall, our study suggests a 'new structural element' in the C-terminal region, i.e. the C-terminal extension, which plays an important role in the oligomerization, subunit exchange dynamics and chaperone function of Hsp16.3.

Role of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from Mycobacterium tuberculosis

“…In the pre-heat treatment assays, it was conjectured that a more favourable secondary structure might aid in improving the chaperone activity of gel-filtered Acr. Our observations indicate that pre-heating the sample might reduce the percentage of β sheets and increase the randomness and disorderliness which corroborates with previous findings (8,10,12). However non-gel-filtered samples did not show any improvement suggesting an analysis of secondary structure that is required to interpret this data.…”

“…High levels of purified Acr were obtained after Histag and gel filtration. Earlier studies have shown Acr to be a nonameric 158 kDa in the form of trimer of trimers, 210 kDa and 193 kDa (4,7,8,10). The activity of A samples was two times less than that of B samples at 37°C.…”

“…Earlier reports on cloning and expression of M. tb. Acr, showed that, Acr molecules can be used for preventing aggregation of the substrates (4)(5)(6)(7)(8). All Acr preparations entailed the use of a gel filtration step to maximize the activity of the protein.…”

“…However, there is no documentation of the oligomeric status of the recombinant protein being used in the assays. It is assumed that Acr is functionally active as a trimer of trimers; or a dodecamer; which was initially reported in 2005 and recently confirmed in 2016 (4,7,8). However, none of these studies mentions the actual oligomeric state of the protein during the assay, the ratio of different size of monomers and about the molecular level interactions except for few reports (9).…”

“…However, few studies have been carried out so far on the interaction at the molecular level in terms of molecules binding to the substrate. Most of the assays in previous studies have used substrates like catalase, citrate synthase, alcohol dehydrogenase and insulin (4,(8)(9)(10). In addition, all the assays reported with insulin B chain were carried out at 25°C or 37°C except for a few studies performed at higher temperatures (10,14).…”