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 (142 STN144 ) 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 (141 RSTN144 ). 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.

中文翻译：

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精氨酸141上的不同突变对Hsp16.3的结构，亚基交换动力学和分子伴侣活性的影响。

Hsp16.3，一种分子伴侣，在宿主内结核分枝杆菌的生长和存活中起着至关重要的作用。我们以前曾报道过，从Hsp16.3的C端延伸（CTE）删除三个氨基酸残基（142 STN144）会触发其结构扰动并增加其伴侣活性，该活性在其整个CTE（141 RSTN144）缺失时达到其顶点。 ）。因此，我们假设Hsp16.3的CTE中的Arg141（R141）和Ser142（S142）可能是维持其天然结构和分子伴侣活性的关键。为了验证该假设，我们生成了两个缺失突变体，其中R141和S142分别缺失（Hsp16.3ΔR​​141和Hsp16.3ΔS142），以及三个替代突变体，其中R141被赖氨酸（Hsp16.3R141K），丙氨酸（Hsp16.3R141A）替代和谷氨酸（Hsp16.3R141E），分别。Hsp16.3ΔS142或Hsp16.3R141K突变体具有天然结构和分子伴侣活性。从CTE中删除R141（Hsp16.3ΔR​​141）会扰乱二级和三级结构，降低亚基交换动力学并降低Hsp16.3的分子伴侣活性。但是，用丙氨酸（Hsp16.3R141A）或谷氨酸（Hsp16.3R141E）取代R141会扰乱其二级和三级结构。令人惊讶的是，R141的这种电荷篡改增强了Hsp16.3的亚基交换动力学和伴侣活性。有趣的是，R141 / S142的缺失或赖氨酸，丙氨酸和谷氨酸的R141取代都不会影响Hsp16.3的寡聚质量/大小。全面的，