The impact of different mutations at arginine141 on the structure, subunit exchange dynamics and chaperone activity of Hsp16.3

Abstract

Hsp16.3, a molecular chaperone, plays a vital role in the growth and survival of Mycobacterium tuberculosis inside the host. We previously reported that deletion of three amino acid residues (142STN144) from C‐terminal extension (CTE) of Hsp16.3 triggers its structural perturbation and increases its chaperone activity, which reaches its apex upon the deletion of its entire CTE (141RSTN144). Thus, we hypothesized that Arg141 (R141) and Ser142 (S142) in the CTE of Hsp16.3 possibly hold the key in maintaining its native‐like structure and chaperone activity. To test this hypothesis, we generated two deletion mutants in which R141 and S142 were deleted individually (Hsp16.3ΔR141 and Hsp16.3ΔS142) and three substitution mutants in which R141 was replaced by lysine (Hsp16.3R141K), alanine (Hsp16.3R141A), and glutamic acid (Hsp16.3R141E), respectively. Hsp16.3ΔS142 or Hsp16.3R141K mutant has native‐like structure and chaperone activity. Deletion of R141 from the CTE (Hsp16.3ΔR141) perturbs the secondary and tertiary structure, lowers the subunit exchange dynamics and decreases the chaperone activity of Hsp16.3. But, the substitution of R141 with alanine (Hsp16.3R141A) or glutamic acid (Hsp16.3R141E) perturbs its secondary and tertiary structure. Surprisingly, such charge tampering of R141 enhances the subunit exchange dynamics and chaperone activity of Hsp16.3. Interestingly, neither the deletion of R141/S142 nor the substitution of R141 with lysine, alanine and glutamic acid affects the oligomeric mass/size of Hsp16.3. Overall, our study suggests that R141 (especially the positive charge on R141) plays a crucial role in maintaining the native‐like structure as well as in regulating subunit exchange dynamics and chaperone activity of Hsp16.3.

Interaction of constituents of MDT regimen for leprosy with Mycobacterium leprae HSP18: impact on its structure and function

“…Enzyme activity of MDH/ADH was measured by taking aliquots from the assay mixture, which was incubated at 43 °C/ 49 °C for 10 min. Enzymatic activity of MDH and ADH was estimated as described previously [29,72,[88][89][90].…”

“…The reported spectra in far-UV CD experiment were the average of five scans. The spectra were further analyzed for secondary structure content by the curve-fitting program CDNN [29,72,[89][90].…”