Abstract
The periplasmic histidine permease of Salmonella typhimurium is composed of a membrane-bound complex and a soluble histidine-binding protein (the periplasmic receptor), HisJ. Liganded receptor interacts with the membrane-bound complex, inducing ATP hydrolysis and substrate translocation. Preliminary evidence had shown a lack of direct correlation between the affinity of HisJ for a ligand and translocation efficiency, suggesting that the precise form of the receptor is important in determining its interaction with the membrane-bound complex. We have investigated the nature of the conformations assumed by HisJ upon binding a variety of ligands by tryptophan fluorescence enhancement, reaction with a closed form-specific monoclonal antibody, and changes in UV absorption spectra. It is demonstrated that although HisJ binds all the ligands and undergoes a conformational change, it assumes measurably different conformations. We also show that the interaction between HisJ and the membrane-bound complex depends on the nature of the ligand. Transport specificity appears to be defined, at least in part, by the conformation of the bound receptor, manifested either by the effect of a given ligand on the closed structure per se, or by the effect of ligand association on the equilibrium constant relating the open and the closed liganded forms.
Highlights
The periplasmic histidine permease of Salmonella typhimurium is composed of a membrane-bound complex and a soluble histidine-binding protein, HisJ
We have investigated the nature of the conformations assumed by HisJ upon binding a variety of ligands by tryptophan fluorescence enhancement, reaction with a closed form-specific monoclonal antibody, and changes in UV absorption spectra
The nature of the liganded forms of HisJ was investigated by four techniques: the fluorescence of the single tryptophan present in HisJ (Trp-130) [15, 27, 44], the interaction of HisJ with the closed form-specific mAb, 9D2 [15, 17], the UV absorption spectrum [24, 26], and cross-linking between the various forms of HisJ and HisQ/M/P [38]
Summary
HisQ/M/P, HisQ/HisM/HisP memdefrayed in part by the payment of page charges. This article must brane-bound complex; ELISA, enzyme-linked immunosorbent assay; be hereby marked “advertisement” in accordance with 18 MOPS, 4-morpholinepropanesulfonic acid; mAb, monoclonal antibody;. Unliganded HisJ (0.4 M, in 10 mM MOPS buffer, pH 7.0) was incubated for 5 min with ligands (0.5, 2.0, 1.0, 100, 100, 100, 1000, and 1000 M for L-histidine, L-arginine, L-lysine, L-azaserine, D-HIPA, N-acetyl-L-histidine, L-histidinol, and D-histidine, respectively), followed by the addition of 9D2 (40 nM) and 1 h incubation at room temperature to permit completion of the association of the mAb with HisJ-ligand complexes. This was followed by addition of 10 M L-[3H]histidine, and dialysis against ligand-free buffer (24 h at 4 °C) to remove free L-[3H]histidine. As suggested above, receptors either assume different overall conformations with different ligands, or that the equilibrium between the open liganded and the closed liganded forms is a function of the ligand structure
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