Detection of a conserved alpha-helix in the kinase-docking region of the aspartate receptor by cysteine and disulfide scanning

Abstract

The transmembrane aspartate receptor of Escherichia coli and Salmonella typhimurium propagates extracellular signals to the cytoplasm, where its cytoplasmic domain regulates the histidine kinase, CheA. Different signaling states of the cytoplasmic domain modulate the kinase autophosphorylation rate over at least a 100-fold range. Biochemical and genetic studies have implicated a specific region of the cytoplasmic domain, termed the signaling subdomain, as the region that transmits regulation from the receptor to the kinase. Here cysteine and disulfide scanning are applied to the N-terminal half of the signaling subdomain to probe its secondary structure, solvent exposure, and protein-protein interactions. The chemical reactivities of the scanned cysteines exhibit the characteristic periodicity of an alpha-helix with distinct solvent-exposed and buried faces. This helix, termed alpha7, ranges approximately from residue 355 through 386. Activity measurements probing the effects of cysteine substitutions in vivo and in vitro reveal that both faces of helix alpha7 are critical for kinase activation, while the buried face is especially critical for kinase down-regulation. Disulfide scanning of the region suggests that helix alpha7 is not in direct contact with its symmetric partner (alpha7') from the other subunit; presently, the structural element that packs against the buried face of the helix remains unidentified. Finally, a novel approach termed "protein interactions by cysteine modification" indicates that the exposed C-terminal face of helix alpha7 provides an essential docking site for the kinase CheA or for the coupling protein CheW.

FIG. 1. A schematic model of the full-length membrane-bound aspartate receptor, illustrating the various domains and subdomains

Cylinders represent helices determined by previous studies employing crystallography (1) and cysteine and disulfide scanning (–36, 41, 42, 57, 58). The two 60-kDa subunits of the homodimer are depicted in white and gray, respectively. Filled circles represent the sites of adaptive methylation on each subunit (49). The open box denotes the region of the signaling subdomain probed by cysteine and disulfide scanning in the current study (residues Thr³⁴⁸ through Ala³⁸⁷).

FIG. 2. Comparison of measured chemical reactivities and calculated solvent exposure in a known α-helix

Cysteine scanning was used to place a cysteine residue at positions 95–103 of helix α2 in the known structure of the periplasmic domain. Each cysteine-containing receptor was incubated with a sulfhydryl-specific probe, either 5-IAF (open circles) or IANBD (open squares) for 5 min at 25 °C. Subsequently, half the reaction was quenched with excess BME, while the other half was denatured with heat and SDS to allow full labeling before quenching. The chemical reactivity parameter is defined as the ratio of receptor labeling in the native sample to the denatured sample, followed by normalization to the highest reactivity observed for each probe. The solvent exposure of the β-carbon at each position, indicated by the filled circles, was calculated from the crystal structure of the periplasmic domain (1) by the method of Richards (71).

FIG. 3. Chemical reactivities of residues T348C through A387C in the signaling subdomain

Using 5-IAF as the probe, the chemical reactivity of each cysteine-containing receptor in isolated E. coli membranes was measured as described in the legend to Fig. 2. Positions with a reactivity of below 0.15, indicated by the lower dashed line, are defined as highly buried. Positions exhibiting a reactivity above 0.35, indicated by the upper dashed line, are classified as solvent-exposed.

FIG. 4. In vivo activity of the engineered cysteine-containing receptors

Engineered receptors were expressed in an E. coli strain lacking the aspartate receptor, and the ability of each receptor to restore chemotaxis up an aspartate gradient was measured by the swarm assay (33, 69). The aspartate-specific swarm rate represents the difference between the chemotactic swarm rates measured on minimal media plates containing and lacking aspartate, respectively. In addition, the indicated rate differences have been normalized to the corresponding difference observed for cells expressing the wild type receptor. Receptors yielding rates below the dashed line (50% of wild type) are classified as inhibitory.

FIG. 5. In vitro activity of the engineered receptors

Isolated E. coli membranes containing a given receptor were mixed with purified cytoplasmic components to reconstitute the receptor-CheW-CheA ternary complex and its phosphorylation target, CheY (12, 13, 33). [³²P]ATP was added to initiate the phosphorylation cascade, and the reaction was quenched after 10 s. The resulting [³²P]phospho-CheY was quantitated and used to determine the rate of phospho-CheY production, which was finally normalized to the wild type rate. Closed circles indicate rates observed in the absence of aspartate, wherein the apo receptor activates kinase activity. Apo receptors that yield rates below the lower dashed line (20% of the wild type rate) are defined as inhibitory, while rates above the upper dashed line (200% of the wild type rate) are denoted superactivated. Open circles and thick lines indicate the rates measured in the presence of saturating aspartate. Under these conditions, the wild type rate is diminished to undetectable levels. Filled triangles denote lock-on cysteine positions at which the cysteine substitution prevents full aspartate-triggered kinase down-regulation.

FIG. 6. The effects of covalent modification at specific surface positions on kinase activation in vitro

The indicated receptors in isolated E. coli membranes were modified with the cysteine-specific probe 5-FM or unmodified, respectively. The in vitro activities of the resulting receptors were measured in the receptor-coupled kinase assay (see legend to Fig. 5). The resulting rates of phospho-CheY formation observed in the absence of aspartate are indicated for each unmodified (filled bars) and modified (open bars) receptor.

FIG. 7. Helical wheel model for positions Ser³⁵⁵ through Arg³⁸⁶ of the signaling subdomains illustrating the measured chemical reactivities and activity effects of the scanned cysteines

A, white rectangles denote positions with high chemical reactivities indicating solvent exposure; black rectangles indicate buried positions yielding low chemical reactivities. The face of the helix found to be largely buried is enclosed by the large box, and residues likely to be charged are noted. Positions in parentheses indicate cysteine substitutions that prevent receptor accumulation in the membrane. B, black ovals highlight lock-on cysteine substitutions that prevent full aspartate-induced kinase down-regulation in the in vitro kinase assay. Small open circles or squares denote cysteine substitutions that retain receptor function in the in vivo chemotaxis swarm assay or in the in vitro receptor-coupled kinase assay, respectively. Small closed circles or squares represent cysteine substitutions that inhibit the ability of the receptor to stimulate activity in the swarm assay or receptor-coupled kinase assay, respectively. Large open ovals denote positions that were implicated in CheW docking by genetic studies (60).

FIG. 8. Model of the signaling domain illustrating helix α7 and the results of the PICM study

Cylinders represent experimentally determined helices α6 and α7 (57). Ovals are shown for other regions of unknown secondary structure, which are connected by loops. Small squares indicate the positions examined in the PICM study. Open squares indicate cysteine positions at which 5-FM attachment maintains at least 50% of the kinase activation observed for the unlabeled receptor. The gray and black squares denote positions at which 5-FM labeling maintains less than 25 or 10% of the relative kinase activation, respectively.