Anti‐terminator protein GlpP regulates gene expression of glycerol uptake operon at post‐transcriptional level in a number of bacteria. By now, the molecular dynamics details of ligand and RNA binding by GlpP are still obscure. In this study, we employed the molecular dynamic (MD) simulation and constructed a functional verification platform of GlpP to resolve these puzzles. By combining molecular docking, MD simulation and alanine scanning mutagenesis, a ligand binding pocket consisting of R14, R104 and R157 was identified. Among these residues with positive charge, R14 was dominant for binding glycerol‐3‐phosphate (G3P). Moreover, the “parallel to crossed” conformational change of the predicted RNA binding region was observed in MD simulation. In this process, the interaction between R104 and E129 was crucial to trigger the conformational change. To further verify this speculation, three ligand independent mutants were obtained by error‐prone PCR. The MD simulation indicated that the conformational change happened in all the three mutants, confirming the “parallel to crossed” conformational change endowed GlpP the activity of binding RNA. In recent years, as a potable biological part, anti‐terminator was more and more widely used to regulate gene expression in metabolic engineering and synthetic biology. The work in this study deepened our understanding to the typical anti‐terminator GlpP, contributing to the further engineering and application of this type of regulator.

中文翻译：

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用于配体和RNA结合的抗终止蛋白GlpP的关键残基和构象变化的探索

抗终止蛋白 GlpP 在许多细菌的转录后水平上调节甘油摄取操纵子的基因表达。到目前为止，GlpP 结合配体和 RNA 的分子动力学细节仍不清楚。在这项研究中，我们采用分子动力学 (MD) 模拟并构建了 GlpP 的功能验证平台来解决这些难题。通过结合分子对接、MD 模拟和丙氨酸扫描诱变，鉴定了由 R14、R104 和 R157 组成的配体结合口袋。在这些带正电荷的残基中，R14 在结合 3-磷酸甘油 (G3P) 方面占主导地位。此外，在 MD 模拟中观察到预测的 RNA 结合区域的“平行交叉”构象变化。在这个过程中，R104 和 E129 之间的相互作用对于触发构象变化至关重要。为了进一步验证这一推测，通过易错 PCR 获得了三个与配体无关的突变体。MD 模拟表明，所有三个突变体都发生了构象变化，证实了“平行交叉”的构象变化赋予了 GlpP 结合 RNA 的活性。近年来，作为一种可饮用的生物部分，抗终止子在代谢工程和合成生物学中被越来越广泛地用于调控基因表达。本研究的工作加深了我们对典型的抗终止子 GlpP 的理解，有助于此类调节剂的进一步工程化和应用。确认“平行于交叉”的构象变化赋予 GlpP 结合 RNA 的活性。近年来，作为一种可饮用的生物部分，抗终止子在代谢工程和合成生物学中被越来越广泛地用于调控基因表达。本研究的工作加深了我们对典型的抗终止子 GlpP 的理解，有助于此类调节剂的进一步工程化和应用。确认“平行于交叉”的构象变化赋予 GlpP 结合 RNA 的活性。近年来，作为一种可饮用的生物部分，抗终止子在代谢工程和合成生物学中被越来越广泛地用于调控基因表达。本研究的工作加深了我们对典型的抗终止子 GlpP 的理解，有助于此类调节剂的进一步工程化和应用。