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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity
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Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

机译：大肠杆菌RNase E催化结构域的膜结合可稳定蛋白质结构并增加RNA底物亲和力

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摘要

RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-mem-brane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular di-chroism difference spectroscopy; the dissociation constant (K_d) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeoli-cus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5-sensor, and DNase I subdo-mains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 ℃, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay.
机译：RNase E在RNA加工和衰变以及大肠杆菌细胞质膜的束缚中起着至关重要的作用。然而，这种膜-蛋白质相互作用的功能仍不清楚。在这里，我们建立了RNase E-膜-膜相互作用的机械作用。重组的RNase E的高度保守的N末端片段（NRne，残基1-499）通过静电相互作用与阴离子磷脂特异性结合。 NRne的膜结合特异性用圆二色差光谱法确定。 NRne与阴离子脂质体结合的解离常数（K_d）为298 nM。大肠杆菌RNase G和RNase E / G同源于系统发生距离较远的Aquifex aeoli-cus，流感嗜血杆菌Rd和Synechocystis sp。被发现是膜结合蛋白。发现NRne及其同系物的静电势是保守的，高度正性的，并分布在一个大表面积上，该表面积涵盖在“大”结构域中鉴定出的四个假定的膜结合区（氨基酸1-400，由RNase H组成，大肠杆菌NRne的S1、5-传感器和DNase I亚域）。使用无脂质体和结合脂质体的NRne和RNA底物BR13和GGG-RNAI进行的体外裂解分析表明，NRne膜结合改变了其酶促活性。圆二色光谱显示在10到60℃之间，与膜结合的NRne没有明显的热致结构变化，并且与膜结合的NRne在冷却后仍保持其正常的裂解活性。因此，NRne膜结合通过稳定蛋白质折叠状态并增加其对底物的结合亲和力，诱导了二级蛋白质结构的变化和酶促活化。我们的结果表明，RNase E膜相互作用增强了RNA加工和衰变的速率。

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