Liganded and Unliganded Receptors Interact with Equal Affinity with the Membrane Complex of Periplasmic Permeases, a Subfamily of Traffic ATPases

Abstract

The histidine-binding protein, HisJ, is the soluble receptor for the periplasmic histidine permease of Salmonella typhimurium. The receptor binds the substrate in the periplasm, interacts with the membrane-bound complex, transmits a transmembrane signal to hydrolyze ATP, and releases the ligand for translocation. HisJ, like other periplasmic receptors, has two lobes that are apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation), burying deeply the ligand. Such receptors are postulated to interact with the membrane-bound complex with high affinity in their liganded conformation, and, upon substrate translocation, to undergo a reduction in affinity and therefore be released. Here we show that in contrast to the current postulate, liganded and unliganded receptors have equal affinity for the membrane-bound complex. The affinity is measured both by chemical cross-linking and co-sedimentation procedures. An ATPase activity assay is also used to demonstrate the interaction of unliganded receptor with the membrane-bound complex. These findings support a new model for the transport mechanism, in which the soluble receptor functions independently of the commonly accepted high-low affinity switch.

Characterization of the Adenosine Triphosphatase Activity of the Periplasmic Histidine Permease, a Traffic ATPase (ABC Transporter)

“…The affinity of LAO for HisQMP 2 and the maximum level of stimulated activity (purified HisQMP 2 ) were determined to be 5.3 M and 1.75 mol/ min/mg, respectively. These values are very similar to those obtained for HisJ (13).…”

“…The histidine permease has been reconstituted into PLS that transport histidine when ATP is trapped internally and HisJ is added externally (6,12). As usually assumed in current working models, the liganded receptor sends a signal that initiates ATP hydrolysis and results in subsequent ligand translocation (13,14).…”

The Binding Specificity of OppA Determines the Selectivity of the Oligopeptide ATP-binding Cassette Transporter

“…Even at an OppA concentration in the assay of only 0.36 M, which corresponds to 0.35 nmol/m 2 of membrane surface, the local concentration of OppA at the membrane could be as high as 100 M. Inhibition of transport activity by unliganded SBP has been observed for the periplasmic binding protein-dependent histidine and maltose ABC transport systems (21,23), and this has been ascribed to competition between unliganded and liganded SBP for the translocator. In the case of the histidine permease, it even was suggested that liganded and unliganded HisJ interact with equal affinity with the translocator complex (24). We observed no inhibition at low peptide concentrations, corresponding to local concentrations of 20 M liganded and 80 M unliganded OppA, which indicates that the affinity of the translocator for liganded OppA is much higher than for unliganded OppA.…”

Crystal Structures of the Choline/Acetylcholine Substrate-binding Protein ChoX from Sinorhizobium meliloti in the Liganded and Unliganded-Closed States

“…Ames et al (46) observed identical affinities for the liganded and ligand-free substrate-binding protein HisJ from the histidine transporter of S. typhimurium. The absence of any structural difference between the liganded-closed and unligandedclosed states of either ChoX (this study) or GGBP (19) raises the question of why a substrate-loaded SBP is capable to more strongly stimulate ATPase activity of the transporter in an in vitro assay than the substrate-free form of the binding protein (47,48).…”