LOCK ON OFF DISULFIDES IDENTIFY THE TRANSMEMBRANE SIGNALING HELIX OF THE ASPARTATE RECEPTOR

The aspartate receptor of the bacterial chemotaxis pathway regulates t he autophosphorylation rate of a cytoplasmic histidine kinase in respo nse to ligand binding, The transmembrane signal, which is transmitted from the periplasmic aspartate-binding domain to the cytoplasmic regul atory domain, is carried by an intramolecular conformational change wi thin the homodimeric receptor structure. The present work uses enginee red cysteines and disulfide bonds to probe the nature of this conforma tional change, focusing in particular on the role of the second transm embrane alpha-helix. Altogether 26 modifications, consisting of 13 cys teine pairs and the corresponding disulfide bonds, have been introduce d into the contacts between the second transmembrane helix and adjacen t helices. The effects of these modifications on the transmembrane sig nal have been quantified by in vitro assays which measure (i) ligand b inding, (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 part icular, 13 of the 26 modifications (6 cysteine pairs and 7 disulfides) significantly increase or decrease aspartate affinity, while 15 of th e 26 modifications (5 cysteine pairs and 10 disulfides) destroy transm embrane kinase regulation, Importantly, 3 of the perturbing disulfides are found to lock the receptor in the ''on'' or ''off' signaling stat e by covalently constraining the second transmembrane helix, demonstra ting that it is possible to use engineered disulfides to lock the sign aling function of a receptor protein. A separate aspect of the study p robes the thermal motions of the second transmembrane helix: 4 disulfi des 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 transmembra ne helix is a mobile signaling element responsible for communicating t he transmembrane signal.