Mycobacterium leprae HSP18 is a small heat shock protein (sHSP). It is a major immunodominant antigen of M. leprae pathogen. Previously, we have reported the existence of two M. leprae HSP18 variants in various leprotic patients. One of the variants has serine at position 52, whereas the other one has proline at the same position. We have also reported that HSP18 having proline at position 52 ( HSP18P 52 ) is a nonameric protein and exhibits chaperone function. However, the structural and functional characterization of wild-type HSP18 having serine at position 52 ( HSP18S 52 ) is yet to be explored. Furthermore, the implications of the S52P mutation on the structure and chaperone function of HSP18 are not well understood. Therefore, we cloned and purified these two HSP18 variants. We found that HSP18S 52 is also a molecular chaperone and an oligomeric protein. Intrinsic tryptophan fluorescence and far-UV CD measurements revealed that the S52P mutation altered the tertiary and secondary structure of HSP18. This point mutation also reduced the oligomeric assembly and decreased the surface hydrophobicity of HSP18, as revealed by HPLC and 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid binding studies, respectively. Mutant protein was less stable against thermal and chemical denaturation and was more susceptible towards tryptic cleavage than wild-type HSP18. HSP18P 52 had lower chaperone function and was less effective in protecting thermal killing of Escherichia coli than HSP18S 52. Taken together, our data suggest that serine 52 is important for the larger oligomerization and chaperone function of HSP18. Because both variants differ in stability and function, they may have different roles in the survival of M. leprae in infected hosts. Abbreviations ACD, a-crystallin domain; bis-ANS, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt; CFU, colony-forming unit; FRET, F€ orster resonance energy transfer; Gu-HCl, guanidine hydrochloride; IPTG, isopropyl thio-b-D-galactoside; MDH, malate dehydrogenase; sHSP, small heat shock protein. Structured digital abstract

Background: Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning M. leprae genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of M. leprae is scanty.

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52nd position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52nd position is observed in β4 strand and Proline in 52nd position is observed in loop. The number of residues contributing α helix at N-terminal region varies in both models. In 18S more number of residues is present in α helix when compared to 18P. The loop regions between β3 and β4 strands of both models vary in number of residues present in it. Number of residues contributing β4 strand in both models vary. β6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation.