Department of Biotechnology, Jaypee Institute of Information Technology, Noida 201309, India.
Department of Biotechnology, Jaypee Institute of Information Technology, Noida 201309, India.
Biochim Biophys Acta Gen Subj. 2022 Jun;1866(6):130130. doi: 10.1016/j.bbagen.2022.130130. Epub 2022 Mar 17.
Mycobacterium tuberculosis (Mtb) isocitrate lyase (ICL) is an established drug target that facilitates Mtb persistence. Unlike other mycobacterial strains, where ICL2 is a single gene product, H37Rv has a split event, resulting in two tandemly coded icls - rv1915 and rv1916. Our recent report on functionality of individual Rv1915 and Rv1916, led to postulate the cooperative role of these proteins in pathogen's survival under nutrient-limiting conditions. This study investigates the possibility of Rv1915 and Rv1916 interacting and forming a complex.
Pull down assay, activity assay, mass spectrometry and site directed mutagenesis was employed to investigate and validate Rv1915-Rv1916 complex formation.
Rv1915 and Rv1916 form a stable complex in vitro, with enhanced ICL/MICL activities as opposed to individual proteins. Further, activities monitored in the presence of acetyl-CoA show significant increase for Rv1916 and the complex but not of Rv0467 and Rv1915Δ90CT. Both full length and truncated Rv1915Δ90CT can form complex, implying the absence of its C-terminal disordered region in complex formation. Further, in silico analysis and site-directed mutagenesis studies reveal Y64 and Y65 to be crucial residues for Rv1915-Rv1916 complex formation.
This study uncovers the association between Rv1915 and Rv1916 and supports the role of acetyl-CoA in escalating the ICL/MICL activities of Rv1916 and Rv1915Δ90CT-Rv1916 complex.
Partitioning of ICL2 into Rv1915 and Rv1916 that associates to form a complex in Mtb H37Rv, suggests its importance in signaling and regulation of metabolic pathway particularly in carbon assimilation.
结核分枝杆菌（Mtb）异柠檬酸裂解酶（ICL）是一个既定的药物靶点，有助于 Mtb 的持续存在。与其他分枝杆菌菌株不同，ICL2 是单个基因产物，而 H37Rv 则发生了分裂事件，导致两个串联编码的 icls - rv1915 和 rv1916。我们最近关于单个 Rv1915 和 Rv1916 功能的报告，导致假设这些蛋白质在病原体在营养有限的条件下生存中的合作作用。本研究调查了 Rv1915 和 Rv1916 相互作用并形成复合物的可能性。
采用下拉测定法、活性测定法、质谱法和定点突变法来研究和验证 Rv1915-Rv1916 复合物的形成。
Rv1915 和 Rv1916 在体外形成稳定的复合物，与单独的蛋白质相比，ICL/MICL 活性增强。此外，在乙酰辅酶 A 存在下监测的活性显示 Rv1916 和复合物的活性显著增加，但 Rv0467 和 Rv1915Δ90CT 的活性没有增加。全长和截断的 Rv1915Δ90CT 都可以形成复合物，这意味着其 C 端无规卷曲区在复合物形成中不存在。此外，计算机分析和定点突变研究表明 Y64 和 Y65 是 Rv1915-Rv1916 复合物形成的关键残基。
本研究揭示了 Rv1915 和 Rv1916 之间的关联，并支持乙酰辅酶 A 在提高 Rv1916 和 Rv1915Δ90CT-Rv1916 复合物的 ICL/MICL 活性中的作用。
结核分枝杆菌 H37Rv 中将 ICL2 分配到 Rv1915 和 Rv1916 中，然后形成复合物，这表明它在信号转导和代谢途径，特别是在碳同化中的调控中具有重要作用。
