Zhou H, McEvoy M M, Lowry D F, Swanson R V, Simon M I, Dahlquist F W
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
Biochemistry. 1996 Jan 16;35(2):433-43. doi: 10.1021/bi951960e.
Multidimensional heteronuclear NMR techniques were applied to study a protein fragment of the histidine autokinase CheA from Escherichia coli. This fragment (CheA1-233) contains the phosphotransfer domain and the CheY-binding domain joined by a linker region. Comparison of chemical shift and NOE cross-peak patterns indicates that the structures of the two domains in CheA1-233 remain nearly the same as in the two individual domain fragments, CheA1-134 and CheA124-257. Relaxation properties of the backbone 15N nuclei were measured to study the rotational correlations of the two domains and properties of the linker region. Dynamics data were analyzed both by an isotropic motional model and an anisotropic motional model. The experimental T1 and T2 values, the derived rotational correlation times, and motional anisotropy are significantly different for the two domains, indicating the two domains reorient independently and the linker region is highly flexible. Dynamics data of CheA1-233 were also compared with those of CheA1-134. Our studies show that flexible domain linkers and extended and flexible terminal polypeptide chains can have significant effects on the motional properties of the adjacent structured regions. These observations suggest a model for the graded regulation of CheA autophosphorylation activity. In this model, the various activity states of the receptor are generated by controlling the access of the mean position of the kinase domain to the phosphotransfer domain. This would then modulate the diffusional encounter rate of the domains and hence activity over a wide and graded range of values.
多维异核核磁共振技术被用于研究来自大肠杆菌的组氨酸自激酶CheA的一个蛋白质片段。这个片段（CheA1-233）包含磷酸转移结构域和通过连接区相连的CheY结合结构域。化学位移和NOE交叉峰模式的比较表明，CheA1-233中两个结构域的结构与两个单独的结构域片段CheA1-134和CheA124-257中的结构几乎相同。测量主链15N核的弛豫特性以研究两个结构域的旋转相关性以及连接区的特性。动力学数据通过各向同性运动模型和各向异性运动模型进行分析。两个结构域的实验T1和T2值、推导的旋转相关时间和运动各向异性有显著差异，表明两个结构域独立重新定向，且连接区高度灵活。还将CheA1-233的动力学数据与CheA1-134的动力学数据进行了比较。我们的研究表明，灵活的结构域连接区以及延伸且灵活的末端多肽链可对相邻结构化区域的运动特性产生显著影响。这些观察结果提出了一个CheA自磷酸化活性分级调节的模型。在这个模型中，受体的各种活性状态是通过控制激酶结构域平均位置与磷酸转移结构域的接近程度产生的。这进而会调节结构域的扩散相遇速率，从而在广泛且分级的数值范围内调节活性。
