Cysteine and disulfide scanning reveals a regulatory alpha-helix in the cytoplasmic domain of the aspartate receptor

Abstract

The transmembrane, homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium controls the chemotactic response to aspartate, an attractant, by regulating the activity of a cytoplasmic histidine kinase. The cytoplasmic domain of the receptor plays a central role in both kinase regulation and sensory adaptation, although its structure and regulatory mechanisms are unknown. The present study utilizes cysteine and disulfide scanning to probe residues Leu-250 through Gln-309, a region that contains the first of two adaptive methylation segments within the cytoplasmic domain. Following the introduction of consecutive cysteine residues by scanning mutagenesis, the measurement of sulfhydryl chemical reactivities reveals an alpha-helical pattern of exposed and buried positions spanning residues 270-309. This detected helix, termed the "first methylation helix," is strongly amphiphilic; its exposed face is highly anionic and possesses three methylation sites, while its buried face is hydrophobic. In vivo and in vitro assays of receptor function indicate that inhibitory cysteine substitutions are most prevalent on the buried face of the first methylation helix, demonstrating that this face is involved in a critical packing interaction. The buried face is further analyzed by disulfide scanning, which reveals three "lock-on" disulfides that covalently trap the receptor in its kinase-activating state. Each of the lock-on disulfides cross-links the buried faces of the two symmetric first methylation helices of the dimer, placing these helices in direct contact at the subunit interface. Comparative sequence analysis of 56 related receptors suggests that the identified helix is a conserved feature of this large receptor family, wherein it is likely to play a general role in adaptation and kinase regulation. Interestingly, the rapid rates and promiscuous nature of disulfide formation reactions within the scanned region reveal that the cytoplasmic domain of the full-length, membrane-bound receptor has a highly dynamic structure. Overall, the results demonstrate that cysteine and disulfide scanning can identify secondary structure elements and functionally important packing interfaces, even in proteins that are inaccessible to other structural methods.

FIG. 1. Schematic secondary structure model for the homodimeric cytoplasmic domain of the aspartate receptor based on the pattern recognition in a sequence alignment (56, 57)

Rectangles indicate the regions predicted to be α-helical, and the sites of regulatory methylation are indicated by the filled circles. The small open squares indicate the sites of point mutations that restore signaling function to a defective receptor possessing the A19K mutation in its first transmembrane helix (58). The current study utilizes cysteine and disulfide scanning to probe the region highlighted by the large box (Leu-250 through Gln-309). Although only one subunit is shown in detail, the two identical sub-units are related by two-fold rotational symmetry.

FIG. 2. Cysteine-scanning analysis of chemical reactivity and solvent exposure in the known helix α2 of the periplasmic domain

Shown is the correlation between the chemical reactivities of cysteine sulfhydryl groups and their solvent exposures determined from the crystal structure. Engineered receptors (5 µm monomer) were labeled with 500 µm 5-iodoacetamidofluorescein for 10 min in 10 mm sodium phosphate, pH 6.5, with HCl, 50 mm NaCl, 50 mm KCl, 1 mm EDTA. While one aliquot was quenched immediately, another was first denatured with SDS to generate the unfolded, fully labeled receptor. The measured chemical reactivity (closed symbols, bold line) was defined as the ratio of the labeling observed for the folded receptor to that of the denatured receptor. Solvent exposure (open symbol, fine line) was calculated by the method of Richards (Ref. ; version 1983) using the crystal structure of the apo-periplasmic domain (32).

FIG. 3. Cysteine-scanning analysis of chemical reactivity in the targeted region of the cytoplasmic domain

The chemical reactivity of each engineered cysteine was determined as described in the legend of Fig. 2, yielding the closed symbols. Highly exposed positions are defined as those possessing a relative chemical reactivity above 0.6 (upper dashed line), while highly buried residues are those exhibiting a chemical reactivity below 0.3 (lower dashed line). Also shown (top) are the predicted extents of the putative helices α5 and α6 within the targeted region (see “Discussion”). The gap in the data (positions 265 and 266) indicates cysteine substitutions that block receptor expression.

FIG. 4. Effect of cysteine substitutions on receptor activity

A, relative rates of aspartate-specific chemotactic swarming in vivo. Plotted is the aspartate-specific chemotaxis swarm rate of cells overexpressing a given cysteine-containing receptor relative to the corresponding rate of cells overexpressing the wild-type receptor. Open circles indicate the two cysteine substitutions that block protein expression. The dashed line is drawn at a swarm rate of 0.3, below which the substitution is referred to as inhibitory. B, relative rates of phospho-CheY production by the reduced, reconstituted receptor-CheW-CheA ternary complex. Reactant concentrations were: 3 µm reduced receptor dimer, 2 µm CheW, 0.25 µm CheA monomer, and 10 µm CheY. The buffer was 50 mm Tris, pH 7.5, with HCl, 50 mm KCl 0.3 mm DTT and 5 mm MgCl₂, and the reaction was initiated by the addition of [γ-³²P]ATP to a final concentration of 0.1 mm. The relative rates use the corresponding rate of the wild-type ternary complex as an activity standard. The upper dashed line is drawn at a relative rate of 1.5, above which the substitution is referred to as superactivating. The lower dashed line is drawn at a relative rate of 0.1, below which the substitution is referred to as highly inhibitory.

FIG. 5. Effect of lock-on disulfides on receptor-mediated kinase regulation

In vitro activity was assayed as described in Fig. 4B. Shown are the relative rates of phospho-CheY production in the absence (open bar) and presence (shaded bar) of 1 mm aspartate. Assays utilized the indicated oxidized receptors in which intersubunit disulfide formation was driven to completion (see “Experimental Procedures”). No disulfide formation was observed for the wild-type receptor, which lacks cysteines.

FIG. 6. Model for cytoplasmic helices α5 and α6, displaying the experimentally determined solvent exposures and activity effects

The helices are shown with the 7-fold periodicity characteristic of coiled-coil or four-helix bundle interactions (–89). A, clustering of the experimentally determined highly buried positions (black boxes) and highly solvent-exposed positions (open boxes) on opposite faces of the putative helices (exposures are defined in Fig. 3). The hydrophobic and charged side chains are also observed to cluster on these same opposite faces, respectively. Basic (+) and acidic (−) side chains are indicated by their charges, while the sites of regulatory methylation are highlighted by filled circles. Two of these methylation sites (positions 295 and 309) are post-translationally converted from glutamine to glutamate by CheB (93). Positions at which cysteine substitution blocks receptor expression are enclosed in parentheses. B, distribution of the experimentally determined sites of inhibitory cysteine substitution. Enclosed by black boxes are the positions where introduction of a cysteine residue inhibits chemotactic swarming in vivo (as defined in Fig. 4). Smaller open squares indicate sites where cysteine substitution superactivates the kinase activity of the ternary complex in vitro, while closed squares indicate sites where cysteine substitution inhibits the ternary complex. Stars denote the positions of lock-on disulfides that constitutively activate the receptor bound kinase.

FIG. 7. Model for the packing of the first methylation helices at the subunit interface

Shown is a view looking from the membrane toward the cytoplasm, in which the experimentally confirmed first methylation helix (α6) is oriented N terminus to C terminus. The putative second methylation helix (α9)is oriented in the antiparallel direction. Positions a and d are the hydrophobic positions of the heptad repeat (see “Discussion”), and the black circles indicate the positions of regulatory methylation. The intersubunit, lock-on disulfide bonds are indicated by the symmetric helix-helix cross-links.