Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
Biochemistry. 2013 Mar 12;52(10):1694-704. doi: 10.1021/bi301189t. Epub 2013 Feb 27.
To identify the crucial residues involved in the self-assembly and function of BfrB, one of the important iron storage proteins of Mycobacterium tuberculosis, we constructed various mutants by employing site-directed mutagenesis. The analysis of mutants led to the identification of "interface hot-spot residues" (R69, L129, and F159) that act as "switch points" for BfrB oligomerization, and our observations show the importance of 4-fold axis residues in assembly formation. Moreover, we demonstrate that single-point mutations Q51A, Q126A, and E135A can enhance the thermal stability of the protein without affecting its assembly. Importantly, a comparative analysis of various mutations revealed that the function of various homologous positions in different ferritins could be at variance; hence, predicting the function of a residue just based on sequence-structure comparisons may not be appropriate. Thus, we report the identification of novel residues in the assembly formation and function of BfrB and show that single-point mutations have a remarkable potential for alteration of multiple properties of ferritins. Besides, "switch residues" or "interface hot spots" identified in this study could also prove to be helpful for the rational design of interfacial inhibitors.
为了确定分枝杆菌重要的铁储存蛋白 BfrB 自我组装和功能所涉及的关键残基，我们通过定点突变构建了各种突变体。突变体分析鉴定出了“界面热点残基”（R69、L129 和 F159），它们作为 BfrB 寡聚化的“开关点”，我们的观察结果表明 4 倍轴残基在组装形成中的重要性。此外，我们证明单点突变 Q51A、Q126A 和 E135A 可以提高蛋白质的热稳定性而不影响其组装。重要的是，对各种突变的比较分析表明，不同铁蛋白中各种同源位置的功能可能存在差异；因此，仅仅基于序列-结构比较来预测残基的功能可能并不合适。因此，我们报告了在 BfrB 的组装形成和功能中鉴定出的新残基，并表明单点突变具有改变铁蛋白多种特性的巨大潜力。此外，本研究中鉴定的“开关残基”或“界面热点”也可能有助于合理设计界面抑制剂。
