THE HISTIDINE-BINDING PROTEIN UNDERGOES CONFORMATIONAL-CHANGES IN THEABSENCE OF LIGAND AS ANALYZED WITH CONFORMATION-SPECIFIC MONOCLONAL-ANTIBODIES

The periplasmic histidine-binding protein, HisJ, and the lysine-, argi nine-, ornithine-binding protein (LAO) are receptors for histidine tra nsport via the histidine permease of Salmonella typhimurium. The recep tors have similar structures, being composed of two lobes held togethe r by two peptide segments, with the ligand-binding site located in a c left between the lobes. The two lobes are far apart in the unliganded structure (open conformation) and are drawn close together in the liga nded structure (closed conformation). The tight binding of the ligand via protein side chains as well as the peptide backbone from both lobe s stabilizes the closed conformation (Oh, B.-H., Pandit, J., Kang, C.- H., Nikaido, K., Gokcen, S., Ames, G. F.-L., and Kim, S.-H. (1993) J. Biol. Chem. 268, 11348-11353; Oh, B.-H., hang C.-H., De Bondt, H., Kim , S.-H., Nikaido, K., Joshi, A., and Ames, G. F.-L. (1994) J. Biol. Ch em. 269, 4135-4143). In this study two conformation-specific monoclona l antibodies (mAbs) that trap the protein in the closed empty form hav e been characterized and used to provide evidence that HisJ can assume the closed empty form in the absence of ligand. Several pieces of evi dence were provided to demonstrate that these mAbs are specific for Hi sJ in the closed form. Histidine improves the interaction of these mAb s with immobilized HisJ. The mAbs inhibit both the exchange and the di ssociation of histidine from HisJ, indicating that they are able to tr ap the protein in the closed liganded form. The characterization of th e epitopes of the conformation-specific mAbs shows that they include r esidues that are located in both lobes and that are far apart in the o pen form but close to each other in the closed form, so that the mAbs must be sensitive to their spatial orientation. Two mAbs that are not conformation-specific according to these criteria were also identified .