Abstract
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.
Highlights
From the +Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, the IlDepartment of Chemistry and Structural Biology, Division of Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720 and the "l,Department of Life Science, Pohang University of Science and Technology, Hyoja-Dong San 31, Pohang, Kyungbuk 790-784, Korea
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
We previously identified monoclonal antibodies that recognize the closed form of HisJ (closed-form-specific mAbs, (Wolf et al, 1994))
Summary
From the +Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, the IlDepartment of Chemistry and Structural Biology, Division of Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720 and the "l,Department of Life Science, Pohang University of Science and Technology, Hyoja-Dong San 31, Pohang, Kyungbuk 790-784, Korea. HisJ has several functions: it binds histidine with high affinity (Kd = 30 na), interacts with the membrane-bound complex, triggers a signaling pathway that results in the hydrolysis of ATP (and/or GTP) and in the release of the ligand from the receptor This series of events results in translocation of the ligand to the cell interior (Prossnitz et al, 1988; Oh et al, 1994b; Bishop et al, 1989; Prossnitz et al, 1989; Joshi et al, 1989; Petronilli and Ames, 1991). There is considerable information on the mechanism of ligand binding by these receptors, little is known about their other functions The goal of this investigation is to gain further understanding of these various functions through a structure/function analysis of HisJ using mutant proteins defective in transport. We discuss the significance of our findings for the mechanism of transport in general
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