Structure/Function Analysis of the Periplasmic Histidine-binding Protein

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.

Mutagenesis and Modeling of the GABAB Receptor Extracellular Domain Support a Venus Flytrap Mechanism for Ligand Binding

“…Accordingly, mutation of these residues may affect K 2 and therefore have opposite effects on the affinities of agonists and antagonists. In agreement with this proposal, amino acids at comparable positions in PBPs such as the histidine-binding protein have been shown to affect the equilibrium constant between the closed and open states (40,41,44). It is interesting to note here that Ser-247 aligns with Ser-73 in LBP and Ser-166 in mGluR1.…”

Spermidine-preferential Uptake System in Escherichia coli

“…Between the domains lies a large cleft that binds the substrate. Furthermore, structure/function analysis of histidine-binding protein was performed with several mutated histidine-binding proteins (32,33). In the case of PotD protein, it was suggested by x-ray crystal structural analysis (13) that 13 amino acids may be involved in the interaction with spermidine.…”

Purification and Characterization of HisP, the ATP-binding Subunit of a Traffic ATPase (ABC Transporter), the Histidine Permease of Salmonella typhimurium

“…Miscellaneous Assays-SDS-PAGE and immunoblotting were performed as described (21), using polyclonal antibody raised against HisP (lot 1618), and quantitating the bands with an Alpha Imager (IS-1000; Alpha Innotech Corp.). Phospholipids, various nucleotides, and ATPase inhibitors were obtained and prepared as described (7).…”