Wolf A, Shaw E W, Oh B H, De Bondt H, Joshi A K, Ames G F
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
J Biol Chem. 1995 Jul 7;270(27):16097-106. doi: 10.1074/jbc.270.27.16097.
The periplasmic histidine-binding protein, HisJ, is a receptor for the histidine permease of Salmonella typhimurium. Receptors of this type are composed of two lobes that are far apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation). The binding of the ligand, in a cleft between the lobes, stabilizes the closed conformation. Such receptors have several functions in transport: interaction with the membrane-bound complex, transmission of a transmembrane signal to hydrolyze ATP, and receiving a signal to open the lobes and release the ligand. In this study the mechanism of action of HisJ was further investigated using mutant proteins defective in ligand binding activity and closed form-specific monoclonal antibodies (Wolf, A., Shaw, E. W., Nikaido, K., and Ames G. F.-L. (1994) J. Biol. Chem. 269, 23051-23058). Y14H is defective in stabilization of the closed form, does not assume the closed empty form, and assumes an altered closed liganded form. T121A and G119R are similar to Y14H, but assume a normal closed liganded form. S72P binds the ligand to the open form, but does not assume a recognizable closed form. S92F is defective in the ability to undergo conformational change and to stabilize the closed form. All other mutant proteins appear to fall within one of these four categories. The biochemical characterization of these mutant proteins agrees with the structural analysis of the protein. We suggest that mutant proteins that do not assume the normal closed form, in addition to their defect in ligand binding, fail to interact with the membrane-bound complex and/or to transmit transmembrane signals.
周质组氨酸结合蛋白HisJ是鼠伤寒沙门氏菌组氨酸通透酶的一种受体。这类受体由两个叶组成，在未结合配体的结构（开放构象）中相距很远，而在结合配体的结构（封闭构象）中靠得很近。配体在叶之间的裂隙中结合，稳定封闭构象。这类受体在转运过程中有多种功能：与膜结合复合物相互作用、传递跨膜信号以水解ATP，以及接收打开叶并释放配体的信号。在本研究中，使用配体结合活性缺陷的突变蛋白和封闭形式特异性单克隆抗体，进一步研究了HisJ的作用机制（Wolf, A., Shaw, E. W., Nikaido, K., and Ames G. F.-L. (1994) J. Biol. Chem. 269, 23051 - 23058）。Y14H在封闭形式的稳定方面存在缺陷，不呈现封闭的空形式，而是呈现改变的封闭配体形式。T121A和G119R与Y14H相似，但呈现正常的封闭配体形式。S72P将配体结合到开放形式，但不呈现可识别的封闭形式。S92F在发生构象变化和稳定封闭形式的能力方面存在缺陷。所有其他突变蛋白似乎都属于这四类中的一类。这些突变蛋白的生化特性与该蛋白的结构分析一致。我们认为，除了在配体结合方面存在缺陷外，不呈现正常封闭形式的突变蛋白无法与膜结合复合物相互作用和/或传递跨膜信号。
