Bacterial chemoreceptors: providing enhanced features to two-component signaling.

Bacteria perform chemotaxis utilizing core two-component signaling systems to which have been added enhanced features of signal amplification, sensory adaptation, molecular memory and high sensitivity over a wide dynamic range. Chemoreceptors are central to the enhancements. These transmembrane homodimers associate in trimers and in clusters of signaling complexes containing from a few to thousands of receptors. Receptor homodimers couple ligand occupancy and adaptational modification to transmembrane signaling. Trimers activate and control the histidine kinase. Clusters enable signal amplification, high sensitivity and adaptational assistance. Homodimer signaling initiates with helical piston sliding that is converted to modulation of competing packing modes of adjacent segments of an extended helical coiled coil. In trimers, signaling and coupling may involve switching between compact and expanded forms.

Analyzing transmembrane chemoreceptors using in vivo disulfide formation between introduced cysteines.

The sulfhydryl chemistry possible at the thiol group of cysteine provides a very useful tool for probing protein structure and function. The power of site-specific mutagenesis makes it possible to use this tool at essentially any position in a polypeptide sequence. The reactivity of introduced cysteines is often assessed in vitro, using purified proteins or cell extracts. However, it can be particularly informative to probe the protein of interest in vivo, in its native cellular environment. Our laboratory has used in vivo approaches extensively in studies of bacterial transmembrane chemoreceptors, particularly by utilizing disulfide formation between pairs of introduced cysteines to learn about structural organization and mechanisms of function. We have concentrated on experimental conditions in which the cellular system of interest remained functional and thus the protein we were characterizing maintained not only its native structure but also its natural interactions. For this reason, our studies of bacterial transmembrane chemoreceptors using disulfide formation in vivo have focused in large part on cysteines separated from the reducing environment of the cell interior, in transmembrane or periplasmic domains. In this chapter, we discuss the applications and limitation of these approaches as well as the details of experimental manipulations and data analysis.

Adaptational modification and ligand occupancy have opposite effects on positioning of the transmembrane signalling helix of a chemoreceptor.

Sensory systems adapt to persistent stimulation. In the transmembrane receptors of bacterial chemotaxis, adaptation is mediated by methylation at specific glutamyl residues in the cytoplasmic domain. Methylation counteracts effects of ligand binding on functional activities of that domain. Both ligand binding and adaptational modification are thought to act through conformational changes. As characterized for Escherichia coli chemoreceptors, a mechanistically crucial feature of the ligand-induced conformational change is piston sliding towards the cytoplasm of a signalling helix in the periplasmic/transmembrane domain. Adaptational modification could counteract this signalling movement by blocking its influence on the cytoplasmic domain or by reversing it. To investigate, we characterized effects of adaptational modification on the position of the signalling helix in chemoreceptor Trg using rates of disulphide formation between introduced cysteines. We utilized an intact cell procedure in which receptors were in their native, functional state. In vivo rates of disulphide formation between diagnostic cysteine pairs spanning a signalling helix interface changed as a function of adaptational modification. Strikingly, those changes were opposite those caused by ligand occupancy for each diagnostic pair tested. This suggests that adaptational modification resets the receptor complex to its null state by reversal of the conformational change generated by ligand binding.

Similarities and differences in interactions of the activity-enhancing chemoreceptor pentapeptide with the two enzymes of adaptational modification.

Sensory adaptation and chemotaxis by Escherichia coli require a specific pentapeptide at the chemoreceptor carboxyl terminus. This sequence binds the two enzymes of receptor adaptational modification, enhancing catalysis, but with different binding features and mechanisms. We investigated the relative importance of each pentapeptide side chain for the two enhancing interactions.

Diagnostic cross-linking of paired cysteine pairs demonstrates homologous structures for two chemoreceptor domains with low sequence identity.

Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg(E) from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the Trg(E) domain was created using the homodimeric, four-helix bundle structure of the Tar(S) domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg(E) periplasmic domain is very similar to the Tar(S) domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.

Carboxyl-terminal extensions beyond the conserved pentapeptide reduce rates of chemoreceptor adaptational modification.

Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors. Specific glutamyl residues are methylated and demethylated in reactions catalyzed by methyltransferase CheR and methylesterase CheB. In the well-characterized chemosensory systems of Escherichia coli and Salmonella spp., efficient modification by either enzyme is dependent on a conserved pentapeptide sequence, NWETF or NWESF, present at the extreme carboxyl terminus of high-abundance chemoreceptors. To what extent is position at the extreme carboxyl terminus important for pentapeptide-mediated enhancement of adaptational modification? Is this position equally important for enhancement of both enzyme activities? To address these questions, we created forms of high-abundance receptor Tsr or Tar carrying one, six, or eight additional amino acids extending beyond the pentapeptide at their carboxyl termini and assayed methylation, demethylation, deamidation, and ability to mediate chemotaxis. In vitro and in vivo, all three carboxyl-terminal extensions reduced pentapeptide-mediated enhancement of rates of adaptational modification. CheB-catalyzed reactions were more affected than CheR-catalyzed reactions. Effects were less severe for the complete sensory system in vivo than for the minimal system of receptor and modification enzymes in vitro. Notably, extended receptors mediated chemotaxis as efficiently as wild-type receptors, providing a striking example of robustness in chemotactic systems. This could reflect compensatory reductions of rates for both modification reactions, mitigation of effects of slower reactions by the intertwined circuitry of signaling and adaptation, or tolerance of a range of reactions rates for adaptational modification. No matter what the mechanism, the observations provide a challenging test for mathematical models of chemotaxis.