Domain organization and flavin adenine dinucleotide-binding determinants in the aerotaxis signal transducer Aer of Escherichia coli
Domain organization and flavin adenine dinucleotide-binding determinants in the aerotaxis signal transducer Aer of Escherichia coli
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DOI：
10.1073/pnas.100118697
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发表时间：
2000-05-23
影响因子：
11.1
通讯作者：
Parkinson, JS
中科院分区：
文献类型：
--
作者：
Bibikov, SI;Barnes, LA;Parkinson, JS
大肠杆菌中的趋气反应由膜换能器Aer介导，Aer是最近鉴定的PAS结构域蛋白超家族的成员，其包括光、氧和氧化还原状态的传感器。对Aer的初步研究表明，它可能利用黄素腺嘌呤二核苷酸（FAD）辅基来监测细胞氧化还原的变化。为了验证这一想法，我们通过His-标签亲和色谱法纯化了月桂基麦芽糖苷溶解的Aer蛋白，并通过高效液相色谱法、质谱法和吸收光谱法显示其非共价地结合FAD。跨越Aer的N-末端290个残基的多肽片段（其包含PAS基序）能够结合FAD。Aer的这一部分与Tsr的鞭毛信号结构域融合，丝氨酸化学感受器，产生了一个功能性的趋气性传感器，表明FAD结合部分的Aer是足够的气敏。除PAS结构域外，Aerotaxis缺陷型错义突变体还确定了在FAD结合中发挥作用的两个区域。这些区域位于锚Aer所需的中央疏水片段的两侧。它们可能直接接触FAD配体或稳定FAD结合口袋。然而，它们在其他细菌的Aer同源物中缺乏序列保守性，表明它们在FAD结合中发挥的作用不那么直接。一个或两个区域可能也发挥重要作用，在传输刺激诱导的构象变化的C-末端鞭毛信号结构域触发aerotactic行为反应。
Aerotactic responses in Escherichia coli are mediated by the membrane transducer Aer, a recently identified member of the superfamily of PAS domain proteins, which includes sensors of light, oxygen, and redox state. Initial studies of Aer suggested that it might use a flavin adenine dinucleotide (FAD) prosthetic group to monitor cellular redox changes. To test this idea, we purified lauryl maltoside-solubilized Aer protein by His-tag affinity chromatography and showed by high performance liquid chromatography, mass spectrometry, and absorbance spectroscopy that it hound FAD noncovalently, Polypeptide fragments spanning the N-terminal 290 residues of Aer, which contains the PAS motif, were able to bind FAD, Fusion of this portion of Aer to the flagellar signaling domain of Tsr, the serine chemoreceptor, yielded a functional aerotaxis transducer, demonstrating that the FAD-binding portion of Aer is sufficient for aerosensing. Aerotaxis-defective missense mutants identified two regions, in addition to the PAS domain, that play roles in FAD binding. Those regions flank a central hydrophobic segment needed to anchor Aer to the cytoplasmic membrane. They might contact the FAD ligand directly or stabilize the FAD-binding pocket. However, their lack of sequence conservation in Aer homologs of other bacteria suggests that they play less direct roles in FAD binding. One or both regions probably also play important roles in transmitting stimulus-induced conformational changes to the C-terminal flagellar signaling domain to trigger aerotactic behavioral responses.