McEvoy M M, Zhou H, Roth A F, Lowry D F, Morrison T B, Kay L E, Dahlquist F W
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
Biochemistry. 1995 Oct 24;34(42):13871-80. doi: 10.1021/bi00042a019.
CheA is the histidine autokinase in the Escherichia coli chemotaxis signal transduction pathway responsible for coupling of signals received by transmembrane receptors to the response regulators CheY and CheB. Here NMR spectroscopy is used to study a 14 kDa fragment of CheA, residues 124-257, that binds the response regulator CheY. Backbone atom resonance assignments were obtained by analysis of 3D HNCACB, 3D CBCA(CO)NH, and HNCO spectra, whereas side-chain assignments were obtained primarily by analysis of 3D H(CCO)NH, 3D C(CO)NH, 3D HCCH-TOCSY, and 3D 1H, 15N TOCSY-HSMQC spectra. NOE cross peak patterns and intensities as well as torsion angle restraints were used to determine the secondary structure, and a low-resolution structure was calculated by hybrid distance-geometry simulated annealing methods. The CheA124-257 fragment consists of four antiparallel beta strands and two helices, arranged in an "open-faced beta-sandwich" motif, as well as two unstructured ends that correspond to domain linkers in the full-length protein. The 15N-1H correlation spectrum of 15N-labeled CheA124-257 bound to unlabeled CheY shows specific localized changes that may correspond to a CheY-binding face on CheA.
CheA是大肠杆菌趋化信号转导途径中的组氨酸自激酶，负责将跨膜受体接收到的信号与应答调节因子CheY和CheB偶联。本文利用核磁共振光谱研究了CheA的一个14 kDa片段（第124 - 257位氨基酸残基），该片段可结合应答调节因子CheY。通过分析三维HNCACB、三维CBCA(CO)NH和HNCO光谱获得主链原子共振归属，而侧链归属主要通过分析三维H(CCO)NH、三维C(CO)NH、三维HCCH - TOCSY和三维1H, 15N TOCSY - HSMQC光谱获得。利用NOE交叉峰模式和强度以及扭转角限制来确定二级结构，并通过混合距离几何模拟退火方法计算低分辨率结构。CheA124 - 257片段由四条反平行β链和两条螺旋组成，排列成“开放式β三明治”基序，以及两个对应于全长蛋白质中结构域连接子的无结构末端。与未标记的CheY结合的15N标记的CheA124 - 257的15N - 1H相关光谱显示出特定的局部变化，这可能对应于CheA上的CheY结合面。
