Butler S L, Falke J J
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Biochemistry. 1998 Jul 28;37(30):10746-56. doi: 10.1021/bi980607g.
The transmembrane aspartate receptor of E. coli and S. typhimurium mediates cellular chemotaxis toward aspartate by regulating the activity of the cytoplasmic histidine kinase, CheA. Ligand binding results in transduction of a conformational signal through the membrane to the cytoplasmic domain where both kinase regulation and adaptation occur. Of particular interest is the linker region, E213 to Q258, which connects and transduces the conformational signal between the cytoplasmic end of the transmembrane signaling helix (alpha 4/TM2) and the major methylation helix of the cytoplasmic domain (alpha 6). This linker is crucial for stable folding and function of the homodimeric receptor. The present study uses cysteine and disulfide scanning mutagenesis to investigate the secondary structure and packing surfaces within the linker region. Chemical reactivity assays reveal that the linker consists of three distinct subdomains: two alpha-helices termed alpha 4 and alpha 5 and, between them, an ordered region of undetermined secondary structure. When cysteine is scanned through the helices, characteristic repeating patterns of solvent exposure and burial are observed. Activity assays, both in vivo and in vitro, indicate that each helix possesses a buried packing face that is crucial for proper receptor function. The interhelical subdomain is at least partially buried and is also crucial for proper receptor function. Disulfide scanning places helix alpha 4 distal to the central axis of the homodimer, while helix alpha 5 is found to lie at the subunit interface. Finally, sequence alignments suggest that all three linker subdomains are highly conserved among the large subfamily of histidine kinase-coupled sensory receptors that possess methylation sites for use in covalent adaptation.
大肠杆菌和鼠伤寒沙门氏菌的跨膜天冬氨酸受体通过调节细胞质组氨酸激酶CheA的活性，介导细胞对天冬氨酸的趋化作用。配体结合导致构象信号通过膜传递到细胞质结构域，在那里发生激酶调节和适应性变化。特别令人感兴趣的是连接区域E213至Q258，它在跨膜信号螺旋（α4/TM2）的细胞质末端与细胞质结构域的主要甲基化螺旋（α6）之间连接并传递构象信号。该连接区域对于同二聚体受体的稳定折叠和功能至关重要。本研究使用半胱氨酸和二硫键扫描诱变来研究连接区域内的二级结构和堆积表面。化学反应性分析表明，该连接区域由三个不同的亚结构域组成：两个称为α4和α5的α螺旋，以及它们之间二级结构不确定的有序区域。当半胱氨酸扫描这些螺旋时，观察到溶剂暴露和埋藏的特征性重复模式。体内和体外活性分析表明，每个螺旋都有一个埋藏的堆积面，这对于受体的正常功能至关重要。螺旋间亚结构域至少部分被埋藏并且对于受体的正常功能也至关重要。二硫键扫描表明α4螺旋位于同二聚体中心轴的远端，而α5螺旋位于亚基界面处。最后，序列比对表明，在具有用于共价适应性的甲基化位点的组氨酸激酶偶联感觉受体的大亚家族中，所有三个连接区域亚结构域都高度保守。
