Department of Biotechnology, National Institute of Technology Warangal, Warangal, Telangana, 506004, India.
Mol Biotechnol. 2021 Mar;63(3):200-220. doi: 10.1007/s12033-020-00293-5. Epub 2021 Jan 9.
The mce1 operon of Mycobacterium tuberculosis, important for lipid metabolism/transport, host cell invasion, modulation of host immune response and pathogenicity, is under the transcriptional control of Mce1R. Hence characterizing Mce1R is an important step for novel anti-tuberculosis drug discovery. The present study reports functional and in silico characterization of Mce1R. In this work, we have computationally modeled the structure of Mce1R and have validated the structure by computational and experimental methods. Mce1R has been shown to harbor the canonical VanR-like structure with a flexible N-terminal 'arm', carrying conserved positively charged residues, most likely involved in the operator DNA binding. The mce1R gene has been cloned, expressed, purified and its DNA-binding activity has been measured in vitro. The K value for Mce1R-operator DNA interaction has been determined to be 0.35 ± 0.02 µM which implies that Mce1R binds to DNA with moderate affinity compared to the other FCD family of regulators. So far, this is the first report for measuring the DNA-binding affinity of any VanR-type protein. Despite significant sequence similarity at the N-terminal domain, the wHTH motif of Mce1R exhibits poor conservancy of amino acid residues, critical for DNA-binding, thus results in moderate DNA-binding affinity. The N-terminal DNA-binding domain is structurally dynamic while the C-terminal domain showed significant stability and such profile of structural dynamics is most likely to be preserved in the structural orthologs of Mce1R. In addition to this, a cavity has been detected in the C-terminal domain of Mce1R which contains a few conserved residues. Comparison with other FCD family of regulators suggests that most of the conserved residues might be critical for binding to specific ligand. The max pKd value and drug score for the cavity are estimated to be 9.04 and 109 respectively suggesting that the cavity represents a suitable target site for novel anti-tuberculosis drug discovery approaches.
结核分枝杆菌 mce1 操纵子对脂质代谢/转运、宿主细胞侵袭、宿主免疫反应调节和致病性至关重要，受 mce1R 的转录调控。因此，鉴定 mce1R 是寻找新型抗结核药物的重要步骤。本研究报告了 mce1R 的功能和计算特性。在这项工作中，我们对 mce1R 进行了结构计算建模，并通过计算和实验方法验证了结构。结果表明，mce1R 具有典型的 VanR 样结构，带有柔性的 N 端“臂”，携带保守的正电荷残基，很可能参与操纵子 DNA 结合。克隆、表达、纯化 mce1R 基因，并在体外测量其 DNA 结合活性。mce1R 与操纵子 DNA 相互作用的 K 值为 0.35 ± 0.02µM，这表明与其他 FCD 家族调节剂相比，mce1R 与 DNA 的结合亲和力适中。到目前为止，这是第一个报告测量任何 VanR 型蛋白 DNA 结合亲和力的报告。尽管在 N 端结构域具有显著的序列相似性，但 mce1R 的 wHTH 基序在 DNA 结合的关键氨基酸残基上保守性较差，因此导致 DNA 结合亲和力适中。N 端 DNA 结合结构域结构动态，而 C 端结构域显示出显著的稳定性，这种结构动力学特征很可能在 mce1R 的结构同源物中得以保留。除此之外，还在 mce1R 的 C 端结构域中检测到一个空腔，其中包含一些保守残基。与其他 FCD 家族调节剂的比较表明，大多数保守残基可能对与特定配体结合至关重要。空腔的最大 pKd 值和药物评分分别估计为 9.04 和 109，这表明空腔是新型抗结核药物发现方法的合适靶标。
