Abstract
The aspartate receptor of the bacterial chemotaxis pathway regulates the autophosphorylation rate of a cytoplasmic histidine kinase in response to ligand binding. The transmembrane signal, which is transmitted from the periplasmic aspartate-binding domain to the cytoplasmic regulatory domain, is carried by an intramolecular conformational change within the homodimeric receptor structure. The present work uses engineered cysteines and disulfide bonds to probe the nature of this conformational change, focusing in particular on the role of the second transmembrane alpha-helix. Altogether 26 modifications, consisting of 13 cysteine pairs and the corresponding disulfide bonds, have been introduced into the contacts between the second transmembrane helix and adjacent helices. The effects of these modifications on the transmembrane signal have been quantified by in vitro assays which measure (i) ligand binding, (ii) receptor-mediated regulation of kinase activity, and (iii) receptor methylation. All three parameters are observed to be highly sensitive to perturbations of the second transmembrane helix. In particular, 13 of the 26 modifications (6 cysteine pairs and 7 disulfides) significantly increase or decrease aspartate affinity, while 15 of the 26 modifications (6 cysteine pairs and 10 disulfides) destroy transmembrane kinase regulation. Importantly, 3 of the perturbing disulfides are found to lock the receptor in the "on" or "off" signaling state by covalently constraining the second transmembrane helix, demonstrating that it is possible to use engineered disulfides to lock the signaling function of a receptor protein. A separate aspect of the study probes the thermal motions of the second transmembrane helix: 4 disulfides designed to trap large amplitude twisting motions are observed to disrupt function but form readily, suggesting that the helix is mobile. Together the results support a model in which the second transmembrane helix is a mobile signaling element responsible for communicating the transmembrane signal.