The histidine-binding protein undergoes conformational changes in the absence of ligand as analyzed with conformation-specific monoclonal antibodies.
The periplasmic histidine-binding protein, HisJ, and the lysine-, arginine-, ornithine-binding protein (LAO) are receptors for histidine transport via the histidine permease of Salmonella typhimurium. The receptors have similar structures, being composed of two lobes held together by two peptide segments, with the ligand-binding site located in a cleft between the lobes. The two lobes are far apart in the unliganded structure (open conformation) and are drawn close together in the liganded structure (closed conformation). The tight binding of the ligand via protein side chains as well as the peptide backbone from both lobes 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., Kang, C.-H., De Bondt, H., Kim, S.-H., Nikaido, K., Joshi, A., and Ames, G. F.-L. (1994) J. Biol. Chem. 269, 4135-4143). In this study two conformation-specific monoclonal antibodies (mAbs) that trap the protein in the closed empty form have been characterized and used to provide evidence that HisJ can assume the closed empty form in the absence of ligand. Several pieces of evidence were provided to demonstrate that these mAbs are specific for HisJ in the closed form. Histidine improves the interaction of these mAbs with immobilized HisJ. The mAbs inhibit both the exchange and the dissociation of histidine from HisJ, indicating that they are able to trap the protein in the closed liganded form. The characterization of the epitopes of the conformation-specific mAbs shows that they include residues that are located in both lobes and that are far apart in the open 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.
Wolf
A
A
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Shaw
E W
EW
Nikaido
K
K
Ames
G F
GF
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D033902:Periplasmic Binding Proteins; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D008024:Ligands; D033902:Periplasmic Binding Proteins; C018963:histidine-binding protein; D006639:Histidine