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.
Highlights
The structures of many periplasmic binding proteins have been resolved, and despite the fact that these proteins do not usually bear sequence homology, their structures are similar [7]
Periplasmic permeases are composed of a soluble receptor and a membrane-bound complex, which translocates the substrate with concomitant ATP hydrolysis
Models for their mechanism of action generally depict the liganded receptor as interacting with high affinity with the membrane-bound complex and stimulating ATP hydrolysis by the ATP-binding subunits, which leads to the discharge and translocation of substrate; the unliganded receptor is released because its affinity for the complex is reduced
Summary
HisQ/M/P, membrane-bound complex; PLS, proteoliposomes; LAO, lysine-, arginine-, ornithine-binding protein; JϳQ, cross-linked product between HisJ and HisQ; PAGE, polyacrylamide gel electrophoresis; Sulfo-SANPAH, sulfosuccinimidyl 6-(4Ј-azido-2Ј-nitrophenylamino)hexanoate; MOPS, 4-morpholinepropanesulfonic acid. We show that unliganded receptor interacts with the membrane complex and does so as well as liganded receptor. We challenge the conventional model and propose a different one in which the operation of the receptor is independent of a high-low switch in affinity. The model we propose is relevant for understanding the mechanism of action of several important eukaryotic membrane receptors that contain extracellular substrate-binding domains that are homologous to periplasmic receptors (14 –16)
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