Danielson M A, Bass R B, Falke J J
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
J Biol Chem. 1997 Dec 26;272(52):32878-88. doi: 10.1074/jbc.272.52.32878.
The transmembrane, homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium controls the chemotactic response to aspartate, an attractant, by regulating the activity of a cytoplasmic histidine kinase. The cytoplasmic domain of the receptor plays a central role in both kinase regulation and sensory adaptation, although its structure and regulatory mechanisms are unknown. The present study utilizes cysteine and disulfide scanning to probe residues Leu-250 through Gln-309, a region that contains the first of two adaptive methylation segments within the cytoplasmic domain. Following the introduction of consecutive cysteine residues by scanning mutagenesis, the measurement of sulfhydryl chemical reactivities reveals an alpha-helical pattern of exposed and buried positions spanning residues 270-309. This detected helix, termed the "first methylation helix," is strongly amphiphilic; its exposed face is highly anionic and possesses three methylation sites, while its buried face is hydrophobic. In vivo and in vitro assays of receptor function indicate that inhibitory cysteine substitutions are most prevalent on the buried face of the first methylation helix, demonstrating that this face is involved in a critical packing interaction. The buried face is further analyzed by disulfide scanning, which reveals three "lock-on" disulfides that covalently trap the receptor in its kinase-activating state. Each of the lock-on disulfides cross-links the buried faces of the two symmetric first methylation helices of the dimer, placing these helices in direct contact at the subunit interface. Comparative sequence analysis of 56 related receptors suggests that the identified helix is a conserved feature of this large receptor family, wherein it is likely to play a general role in adaptation and kinase regulation. Interestingly, the rapid rates and promiscuous nature of disulfide formation reactions within the scanned region reveal that the cytoplasmic domain of the full-length, membrane-bound receptor has a highly dynamic structure. Overall, the results demonstrate that cysteine and disulfide scanning can identify secondary structure elements and functionally important packing interfaces, even in proteins that are inaccessible to other structural methods.
大肠杆菌和鼠伤寒沙门氏菌的跨膜同型二聚天冬氨酸受体，通过调节细胞质组氨酸激酶的活性，控制对吸引剂天冬氨酸的趋化反应。受体的细胞质结构域在激酶调节和感觉适应中都起着核心作用，尽管其结构和调节机制尚不清楚。本研究利用半胱氨酸和二硫键扫描来探测250位亮氨酸至309位谷氨酰胺的残基，该区域包含细胞质结构域内两个适应性甲基化片段中的第一个。通过扫描诱变引入连续的半胱氨酸残基后，巯基化学反应性的测量揭示了270 - 309位残基暴露和埋藏位置的α螺旋模式。这个检测到的螺旋，称为“第一个甲基化螺旋”，具有很强的两亲性；其暴露面高度带负电并拥有三个甲基化位点，而其埋藏面是疏水的。受体功能的体内和体外测定表明，抑制性半胱氨酸取代在第一个甲基化螺旋的埋藏面上最为普遍，表明该面参与了关键的堆积相互作用。通过二硫键扫描对埋藏面进行进一步分析，发现三个“锁定”二硫键，它们将受体共价捕获在其激酶激活状态。每个锁定二硫键交联二聚体两个对称的第一个甲基化螺旋的埋藏面，使这些螺旋在亚基界面直接接触。对56种相关受体的比较序列分析表明，所鉴定的螺旋是这个大受体家族的一个保守特征，其中它可能在适应和激酶调节中起普遍作用。有趣的是，扫描区域内二硫键形成反应的快速速率和混杂性质表明，全长膜结合受体的细胞质结构域具有高度动态的结构。总体而言，结果表明半胱氨酸和二硫键扫描可以识别二级结构元件和功能上重要的堆积界面，即使在其他结构方法无法触及的蛋白质中也是如此。
