Differential backbone dynamics of companion helices in the extended helical coiled-coil domain of a bacterial chemoreceptor
Differential backbone dynamics of companion helices in the extended helical coiled-coil domain of a bacterial chemoreceptor
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DOI：
10.1002/pro.2767
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发表时间：
2015-11-01
期刊：
影响因子：
8
通讯作者：
Hazelbauer, Gerald L.
中科院分区：
文献类型：
--
作者：
Bartelli, Nicholas L.;Hazelbauer, Gerald L.
细菌跨膜化学感受器的胞质结构域在很大程度上是扩展的四螺旋卷曲。之前的观察表明，该结构域在结构上是动态的。我们利用定点自旋标记和电子顺磁共振(EPR)谱直接探索了大肠杆菌跨膜化学受体Tar这一结构域的骨架动力学。自旋标记被放置在暴露在溶剂中的螺旋面上，因为这些位置的EPR谱主要反映多肽骨架的运动。我们获得了自旋标记的完整的受体同源二聚体的光谱，这些二聚体溶解在洗涤剂中或插入到纳米盘中天然的大肠杆菌脂质双层中，表征了分布在整个细胞质结构域及其螺旋发夹的两个螺旋上的16个位置，一个氨基末端通向膜的远端紧转角(N-螺旋)，另一个羧基末端(C-螺旋)。洗涤剂的增溶作用增加了大部分结构域的骨架动力学，表明增溶时受体活性的丧失反映了广泛的不稳定。对于任何一种情况下的受体，我们都观察到了N-螺旋和C-螺旋之间的意外差异。对于双层插入的受体，膜-远端蛋白质相互作用区和整个C-螺旋的EPR光谱是结构良好的螺旋的典型特征。相反，大约三分之二的N-螺旋，从它的起源作为膜-近端HAMP结构域的AS-2螺旋到膜-远端蛋白质相互作用区的开始，光谱具有显著的移动性成分，通过光谱去卷积估计平均约为15%。微分螺旋动力学提出了一种具有一对核心支架螺旋和两个更具活力的伙伴螺旋的四螺旋束组织。这种新观察到的化学感受器结构特征可能与受体功能有关。
Cytoplasmic domains of transmembrane bacterial chemoreceptors are largely extended four-helix coiled coils. Previous observations suggested the domain was structurally dynamic. We probed directly backbone dynamics of this domain of the transmembrane chemoreceptor Tar from Escherichia coli using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Spin labels were positioned on solvent-exposed helical faces because EPR spectra for such positions reflect primarily polypeptide backbone movements. We acquired spectra for spin-labeled, intact receptor homodimers solubilized in detergent or inserted into native E. coli lipid bilayers in Nanodiscs, characterizing 16 positions distributed throughout the cytoplasmic domain and on both helices of its helical hairpins, one amino terminal to the membrane-distal tight turn (N-helix), and the other carboxyl terminal (C-helix). Detergent solubilization increased backbone dynamics for much of the domain, suggesting that loss of receptor activities upon solubilization reflects wide-spread destabilization. For receptors in either condition, we observed an unanticipated difference between the N- and C-helices. For bilayer-inserted receptors, EPR spectra from sites in the membrane-distal protein-interaction region and throughout the C-helix were typical of well-structured helices. In contrast, for approximately two-thirds of the N-helix, from its origin as the AS-2 helix of the membrane-proximal HAMP domain to the beginning of the membrane-distal protein-interaction region, spectra had a significantly mobile component, estimated by spectral deconvolution to average approximately 15%. Differential helical dynamics suggests a four-helix bundle organization with a pair of core scaffold helices and two more dynamic partner helices. This newly observed feature of chemoreceptor structure could be involved in receptor function.