A S52P mutation in the ‘α‐crystallin domain’ of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function

Abstract

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

Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

“…To confirm whether changes in the DTNB assay were due to the alteration in the available free thiol moieties of cysteine residues, we performed the assay using the cysteine-lacking Mycobacterium leprae HSP18, which we have previously cloned and purified. 24 We did not observe a change in the absorbance value at 412 nm with time for HSP18. Collectively, these results confirmed that acetylation of γD-crystallin altered the microenvironment of cysteine residues in a manner that rendered them inaccessible for reaction with DTNB.…”

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

“…We also demonstrated that thermally stressed MDH and ADH retained their enzymatic activity to a larger extent in the presence of C-terminal-truncated mutants (except Hsp16.3DC1 and Hsp16.3DC2) than wild-type Hsp16.3. The surface hydrophobicity of Hsp16.3 as well as other sHsps is mostly probed by bis-ANS, a hydrophobic fluorophore [1,9,33,[45][46][47]. Among these, surface hydrophobicity is believed to be one of the important factors behind the modulation of the chaperone function of different sHsps under various conditions [9,33,45,46].…”

“…The reported spectra were the average of five scans [45,47]. Spectra were collected from 200 to 260 nm using a rectangular quartz cell with 1 mm path length.…”

“…The flow rate was maintained at 0.5 mLÁmin À1 for all the measurements [45,47]. After the column was equilibrated with 50 mM phosphate buffer (pH 7.5), 50 lL proteins (0.1-1.0 mgÁmL À1 in the equilibrium buffer) was injected into the column.…”

“…Insulin aggregation was initiated by adding freshly prepared DTT to a final concentration of 20 mM and light scattering at 400 nm was monitored for 1 h in the kinetic mode at 25°C [45,47]. Insulin aggregation was initiated by adding freshly prepared DTT to a final concentration of 20 mM and light scattering at 400 nm was monitored for 1 h in the kinetic mode at 25°C [45,47].…”