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Gerald L Hazelbauer

Publications and source records attributed to Gerald L Hazelbauer.

14 recordsLinked to original sources
PubMed2006

Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties.

Bacterial chemoreceptors are transmembrane homodimers that can form trimers, higher order arrays, and extended clusters as part of signaling complexes. Interactions of dimers in oligomers are thought to confer cooperativity and cross-receptor influences as well as a 35-fold gain between ligand binding and altered kinase activity. In addition, higher order interactions among dimers are necessary for the observed patterns of assistance in adaptational modification among different receptors. Elucidating mechanisms underlying these properties will require defining which receptor functions can be performed by dimers and which require specific higher order interactions. However, such an assignment has not been possible. Here, we used Nanodiscs, an emerging technology for manipulating membrane proteins, to prepare small particles of lipid bilayer containing one or only a few chemoreceptor dimers. We found that receptor dimers isolated in individual Nanodiscs were readily modified, bound ligand, and performed transmembrane signaling. However, they were hardly able to activate the chemotaxis histidine kinase. Instead, maximal activation and thus full-range control of kinase occurred preferentially in discs containing approximately three chemoreceptor dimers. The sharp dependence of kinase activation on this number of receptors per dimer implies that the core structural unit of kinase activation and control is a trimer of dimers. Thus, our observations demonstrate that chemoreceptor transmembrane signaling does not require oligomeric organization beyond homodimers and implicate a trimer of dimers as the unit of downstream signaling.

Dimerization↗
PubMed2006

The carboxyl-terminal linker is important for chemoreceptor function.

Sensory adaptation in bacterial chemotaxis is mediated by chemoreceptor methylation and demethylation. In Escherichia coli, methyltransferase CheR and methylesterase CheB bind both substrate sites and a carboxyl-terminal pentapeptide sequence carried by certain receptors. Pentapeptide binding enhances enzyme action, an enhancement required for effective adaptation and chemotaxis. Pentapeptides are linked to the conserved body of chemoreceptors through a notably variable sequence of 30-35 residues. We created nested deletions from the distal end of this linker in chemoreceptor Tar. Chemotaxis was eliminated by deletion of 20-40 residues and reduced by shorter deletions. This did not reflect generalized disruption, because all but the most extremely truncated receptors activated kinase, were substrates for adaptational modification and performed transmembrane signalling. In contrast, linker truncations reduced rates of adaptational modification in parallel with chemotaxis. We concluded the linker is important for chemotaxis because of its role in adaptational modification. Effects of linker truncations on CheR binding to receptor-borne pentapeptide implied linker (i) makes pentapeptide available to modification enzymes by separation from the helical receptor body, and (ii) is a flexible arm allowing dual binding of enzyme to pentapeptide and modification site. The data suggest linker and the helix from which it emerges are structurally dynamic.

Amino Acid Sequence↗
PubMed2006

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.

Cysteine↗
PubMed2006

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.

Binding Sites↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

Clustering requires modified methyl-accepting sites in low-abundance but not high-abundance chemoreceptors of Escherichia coli.

Chemotaxis signalling complexes of Escherichia coli, composed of chemoreceptors, CheA and CheW, form clusters located predominantly at cell poles. As the only kind of receptor in a cell, high-abundance receptors are polar and clustered whereas low-abundance chemoreceptors are polar but largely unclustered. We found that clustering was a function of the cytoplasmic, carboxyl-terminal domain and that effective clustering was conferred on low-abundance receptors by addition of the approximately 20-residue sequence from the carboxyl terminus of either high-abundance receptor. These sequences are different but share a carboxyl-terminal pentapeptide that enhances adaptational covalent modification and allows a physiological balance between modified and unmodified methyl-accepting sites, implying that receptor modification might influence clustering. Thus we investigated directly effects of modification state on chemoreceptor clustering. As the sole receptor type in a cell, low-abundance receptors were clustered only if modified, but high-abundance receptors were clustered independent of extent of modification. This difference could mean that the two receptor types are fundamentally different or that they are poised at different positions in the same conformational equilibrium. Notably, no receptor perturbation we tested altered a predominant location at cell poles, emphasizing a distinction between determinants of clustering and polar localization.

Amino Acid Sequence↗
PubMed2005

Adaptational assistance in clusters of bacterial chemoreceptors.

Sensory adaptation of low-abundance chemoreceptors in Escherichia coli requires assistance from high-abundance receptors, because only high-abundance receptors carry the carboxyl-terminal pentapeptide sequence NWETF that enhances adaptational covalent modification. Using membrane vesicles containing both high-abundance receptor Tar and low-abundance receptor Trg, we observed effective assistance in vitro for all three adaptational modifications: methylation, demethylation and deamidation. These results demonstrated that adaptational assistance involves not only the previously documented assistance for methylation but also assistance for the two CheB-catalysed reactions. We determined rates of assisted methylation and demethylation at many ratios of assisting to assisted receptor. Analysis by a model of assistance indicated one Tar dimer could assist seven Trg dimers in methylation or five in demethylation, defining assistance neighbourhoods. These neighbourhoods were larger than a trimer of homodimers, required only receptors and were minimally affected by formation of signalling complexes. Time courses of assisted Trg methylation in membranes with low amounts of Tar showed that assisting receptors did not diffuse beyond initial neighbourhoods for at least two hours. Taken together, these observations indicate that chemoreceptors can form stable neighbourhoods larger than trimers in the absence of other chemotaxis proteins. Such interactions are likely to occur in natural receptor clusters in vivo.

Adaptation, Physiological↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2004

Accessibility of introduced cysteines in chemoreceptor transmembrane helices reveals boundaries interior to bracketing charged residues.

Two hydrophobic sequences, 24 and 30 residues long, identify the membrane-spanning segments of chemoreceptor Trg from Escherichia coli. As in other related chemoreceptors, these helical sequences are longer than the minimum necessary for an alpha-helix to span the hydrocarbon region of a biological membrane. Thus, the specific positioning of the segments relative to the hydrophobic part of the membrane cannot be deduced from sequence alone. With the aim of defining the positioning for Trg experimentally, we determined accessibility of a hydrophilic sulfhydryl reagent to cysteines introduced at each position within and immediately outside the two hydrophobic sequences. For both sequences, there was a specific region of uniformly low accessibility, bracketed by regions of substantial accessibility. The two low-accessibility regions were each 19 residues long and were in register in the three-dimensional organization of the transmembrane domain deduced from independent data. None of the four hydrophobic-hydrophilic boundaries for these two membrane-embedded sequences occurred at a charged residue. Instead, they were displaced one to seven residues internal to the charged side chains bracketing the extended hydrophobic sequences. Many hydrophobic sequences, known or predicted to be membrane-spanning, are longer than the minimum necessary helical length, but precise membrane boundaries are known for very few. The cysteine-accessibility approach provides an experimental strategy for determining those boundaries that could be widely applicable.

Amino Acid Sequence↗
PubMed2004

Cellular stoichiometry of the components of the chemotaxis signaling complex.

The chemotactic sensory system of Escherichia coli comprises membrane-embedded chemoreceptors and six soluble chemotaxis (Che) proteins. These components form signaling complexes that mediate sensory excitation and adaptation. Previous determinations of cellular content of individual components provided differing and apparently conflicting values. We used quantitative immunoblotting to perform comprehensive determinations of cellular amounts of all components in two E. coli strains considered wild type for chemotaxis, grown in rich and minimal media. Cellular amounts varied up to 10-fold, but ratios between proteins varied no more than 30%. Thus, cellular stoichiometries were almost constant as amounts varied substantially. Calculations using those cellular stoichiometries and values for in vivo proportions of core components in complexes yielded an in vivo stoichiometry for core complexes of 3.4 receptor dimers and 1.6 CheW monomers for each CheA dimer and 2.4 CheY, 0.5 CheZ dimers, 0.08 CheB, and 0.05 CheR per complex. The values suggest a core unit of a trimer of chemoreceptor dimers, a dimer (or two monomers) of kinase CheA, and two CheW. These components may interact in extended arrays and, thus, stoichiometries could be nonintegral. In any case, cellular stoichiometries indicate that CheY could be bound to all signaling complexes and this binding would recruit essentially the entire cellular complement of unphosphorylated CheY, and also that phosphatase CheZ, methylesterase CheB, and methyltransferase CheR would be present at 1 per 2, per 14, and per 20 core complexes, respectively. These characteristic ratios will be important in quantitative treatments of chemotaxis, both experimental and theoretical.

Bacterial Proteins↗
PubMed2002

Allosteric enhancement of adaptational demethylation by a carboxyl-terminal sequence on chemoreceptors.

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 Escherichia coli and Salmonella enterica serovar typhimurium, efficient adaptational modification by either enzyme is dependent on a conserved pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from the sites of modification. For CheR-catalyzed methylation, previous work demonstrated that this sequence acts as a high affinity docking site, enhancing methylation by increasing enzyme concentration near methyl-accepting glutamates. We investigated pentapeptide-mediated enhancement of CheB-catalyzed demethylation and found it occurred by a distinctly different mechanism. Assays of binding between CheB and the pentapeptide sequence showed that it was too weak to have a significant effect on local enzyme concentration. Kinetic analyses revealed that interaction of the sequence and the methylesterase enhanced the rate constant of demethylation not the Michaelis constant. This allosteric activation occurred if the sequence was attached to chemoreceptor, but hardly at all if it was present as an isolated peptide. In addition, free peptide inhibited demethylation of the native receptor carrying the pentapeptide sequence at its carboxyl terminus. These observations imply that the allosteric change is transmitted through the protein substrate, not the enzyme.

Allosteric Regulation↗
PubMed2002

Site-directed spin labeling of a bacterial chemoreceptor reveals a dynamic, loosely packed transmembrane domain.

We used site-directed spin labeling and electron paramagnetic resonance spectroscopy to investigate dynamics and helical packing in the four-helix transmembrane domain of the homodimeric bacterial chemoreceptor Trg. We focused on the first transmembrane helix, TM1, particularly on the nine-residue sequence nearest the periplasm, because patterns of disulfide formation between introduced cysteines had identified that segment as the region of closest approach among neighboring transmembrane helices. Along this sequence, mobility and accessibility of the introduced spin label were characteristic of loosely packed or solvent-exposed side chains. This was also the case for eight additional positions around the circumference and along the length of TM1. For the continuous nine-residue sequence near the periplasm, mobility and accessibility varied only modestly as a function of position. We conclude that side chains of TM1 that face the interior of the four-helix domain interact with neighboring helices but dynamic movement results in loose packing. Compared to transmembrane segments of other membrane proteins reconstituted into lipid bilayers and characterized by site-directed spin labeling, TM1 of chemoreceptor Trg is the most dynamic and loosely packed. A dynamic, loosely packed chemoreceptor domain can account for many experimental observations about the transmembrane domains of chemoreceptors.

Diffusion↗
PubMed2002

Modeling the transmembrane domain of bacterial chemoreceptors.

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.

Amino Acid Substitution↗