Lock on/off disulfides identify the transmembrane signaling helix of the aspartate receptor

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.

Fig. 1. Schematic structure of the aspartate receptor, including the locations of the engineered cysteines

A, top view of the periplasmic α-helices, looking down their long axes toward the cytoplasm (Milburn et al., 1991). The periplasmic domain consists of two subunits distinguished by shading and primes, each forming a four-helix bundle. The α1 and α1′ helices at the subunit interface form a coiled-coil pair represented by 7-fold helical wheels (Scott et al., 1993), while the remaining standard helices are shown as 18-fold helical wheels. Positions of the engineered cysteines on helices α1 and α4 are indicated; those designed to trap twisting motions of the α4/TM2 helix are underlined. B, model for the packing of the transmembrane helices (Scott and Stoddard, 1994; Pakula and Simon, 1992; Lynch and Koshland, 1991; Falke et al., 1988), illustrating the positions of engineered cysteines on TM1 and TM2. C, schematic representation of the periplasmic and transmembrane helices of each subunit, illustrating the positions of the engineered cysteine pairs placed at the interfaces of helix α4/TM2 with helices α1/TM1 and α3. For simplicity, the cysteine pairs designed to trap α4/TM2 twisting motions are omitted. Given the 2-fold symmetry of the homodimer, both subunits possess the same cysteine pair or disulfide bond. Symbols indicate the degree of motion required for disulfide formation between a given cysteine pair: no motion required (filled square); ≤ 1.5-Å translation required (open square); undetermined (open circle).

Fig. 2. Effect of engineered cysteines and disulfides on the in vitro phosphorylation reaction

Shown is the time course of CheY phosphorylation by the reconstituted receptor-kinase complex, illustrating the activities of the wild-type receptor and three classes of engineered receptors. Isolated E. coli membranes containing the wild-type (WT) and indicated engineered cysteine receptors were reduced (red) to eliminate preexisting disulfide bonds, or oxidized (ox) to drive disulfide bond formation linking engineered cysteine pairs. Subsequently, purified CheA, CheW, and CheY were added to reconstitute the receptor-kinase complex, and the effects of 1 mM aspartate (Asp) on CheY phosphorylation were quantitated (see “Experimental Procedures”). The illustrated reactions are representative of the wild-type receptor (A), or receptors in which an engineered disulfide retains measurable transmembrane kinase regulation (B), locks transmembrane kinase regulation in the off state (C), or locks transmembrane kinase regulation in the on state (D).

Fig. 3. Effects of engineered cysteines and disulfides on the in vitro phosphorylation and methylation reactions: summary

The left panels plot the maximum initial phosphorylation and methylation rates produced by each engineered receptor in its reduced and oxidized states, relative to the activity of the wild-type receptor. The maximum rate of CheY phosphorylation or receptor methylation was obtained in the absence or presence of ligand, respectively. The right panels summarize the ability of aspartate (1.0 mM) to down-regulate CheY phosphorylation, or stimulate receptor methylation, where each aspartate effect is relative to reactions possessing the wild-type receptor. (See Methods and Tables IV and V for further details.)

Fig. 4. Locations of engineered disulfides which retain, restore, or lock the transmembrane signal

Shown is a schematic model of the periplasmic and transmembrane regions of the receptor dimer (note that perspectives have been altered for clarity). The indicated disulfide bonds retain or restore aspartate-triggered transmembrane regulation of kinase activity (solid bar; ≥20% native) or lock the receptor in the on (open bar) or off (stippled bar) signaling state. Formation of a disulfide between the remaining cysteine pairs essentially destroys transmembrane kinase regulation (fine bars; ≤10% native). Only disulfides located at the interfaces of adjacent helical faces are shown; for simplicity, disulfides are shown in just one of the two symmetric subunits, and disulfides designed to trap α4/TM2 twisting motions are omitted. The model proposes that the transmembrane signal originating in the ligand-binding site is generated by an undefined movement of the α4/TM2 helix relative to the subunit interface, which communicates the ligand-binding event to the cytoplasmic domain and its associated histidine kinase.