Significance of a Posttranslational Modification of the PilA Protein of Geobacter sulfurreducens for Surface Attachment, Biofilm Formation, and Growth on Insoluble Extracellular Electron Acceptors.

Geobacter sulfurreducens, an anaerobic metal-reducing bacterium, possesses type IV pili. These pili are intrinsic structural elements in biofilm formation and, together with a number of c-type cytochromes, are thought to serve as conductive nanowires enabling long-range electron transfer (ET) to metal oxides and graphite anodes. Here, we report that a posttranslational modification of a nonconserved amino acid residue within the PilA protein, the structural subunit of the type IV pili, is crucial for growth on insoluble extracellular electron acceptors. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the secreted PilA protein revealed a posttranslational modification of tyrosine-32 with a moiety of a mass consistent with a glycerophosphate group. Mutating this tyrosine into a phenylalanine inhibited cell growth with Fe(III) oxides as the sole electron acceptor. In addition, this amino acid substitution severely diminished biofilm formation on graphite surfaces and impaired current output in microbial fuel cells. These results demonstrate that the capability to attach to insoluble electron acceptors plays a crucial role for the cells' ability to utilize them. The work suggests that glycerophosphate modification of Y32 is a key factor contributing to the surface charge of type IV pili, influencing the adhesion of Geobacter to specific surfaces.IMPORTANCE Type IV pili are bacterial appendages that function in cell adhesion, virulence, twitching motility, and long-range electron transfer (ET) from bacterial cells to insoluble extracellular electron acceptors. The mechanism and role of type IV pili for ET in Geobacter sulfurreducens is still a subject of research. In this study, we identified a posttranslational modification of the major G. sulfurreducens type IV pilin, suggested to be a glycerophosphate moiety. We show that a mutant in which the glycerophosphate-modified tyrosine-32 is replaced with a phenylalanine has reduced abilities for ET and biofilm formation compared with those of the wild type. The results show the importance of the glycerophosphate-modified tyrosine for surface attachment and electron transfer in electrode- or Fe(III)-respiring G. sulfurreducens cells.

Osmotically-induced tension and the binding of N-BAR protein to lipid vesicles.

The binding affinity of a curvature-sensing protein domain (N-BAR) is measured as a function of applied osmotic stress while the membrane curvature is nearly constant. Varying the osmotic stress allows us to control membrane tension, which provides a probe of the mechanism of binding. We study the N-BAR domain of the Drosophila amphiphysin and monitor its binding on 50 nm-radius vesicles composed of 90 mol% DOPC and 10 mol% PIP. We find that the bound fraction of N-BAR is enhanced by a factor of approximately 6.5 when the tension increases from zero to 2.6 mN m(-1). This tension-induced response can be explained by the hydrophobic insertion mechanism. From the data we extract a hydrophobic domain area that is consistent with known structure. These results indicate that membrane stress and strain could play a major role in the previously reported curvature-affinity of N-BAR.

Hydrogen exchange differences between chemoreceptor signaling complexes localize to functionally important subdomains.

The goal of understanding mechanisms of transmembrane signaling, one of many key life processes mediated by membrane proteins, has motivated numerous studies of bacterial chemotaxis receptors. Ligand binding to the receptor causes a piston motion of an α helix in the periplasmic and transmembrane domains, but it is unclear how the signal is then propagated through the cytoplasmic domain to control the activity of the associated kinase CheA. Recent proposals suggest that signaling in the cytoplasmic domain involves opposing changes in dynamics in different subdomains. However, it has been difficult to measure dynamics within the functional system, consisting of extended arrays of receptor complexes with two other proteins, CheA and CheW. We have combined hydrogen exchange mass spectrometry with vesicle template assembly of functional complexes of the receptor cytoplasmic domain to reveal that there are significant signaling-associated changes in exchange, and these changes localize to key regions of the receptor involved in the excitation and adaptation responses. The methylation subdomain exhibits complex changes that include slower hydrogen exchange in complexes in a kinase-activating state, which may be partially consistent with proposals that this subdomain is stabilized in this state. The signaling subdomain exhibits significant protection from hydrogen exchange in complexes in a kinase-activating state, suggesting a tighter and/or larger interaction interface with CheA and CheW in this state. These first measurements of the stability of protein subdomains within functional signaling complexes demonstrate the promise of this approach for measuring functionally important protein dynamics within the various physiologically relevant states of multiprotein complexes.

Hydrogen exchange mass spectrometry of functional membrane-bound chemotaxis receptor complexes.

The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (∼2 Å) piston displacement of one helix of the periplasmic and transmembrane domains toward the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) measurements of global exchange of the CF demonstrate that the CF exhibits significantly slower exchange in functional complexes than in solution. Because the exchange rates in functional complexes are comparable to those of other proteins with similar structures, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system.

Membrane association of a protein increases the rate, extent, and specificity of chemical cross-linking.

Many cellular processes involve interactions between membrane-associated proteins, and those interactions are enhanced by membrane association. We have used cross-linking reactions to compare the extent and specificity of protein interactions in solution versus on a membrane surface. Cysteine mutants of a soluble cytoplasmic fragment (CF) of the aspartate receptor, a transmembrane receptor involved in bacterial chemotaxis, are used in disulfide bond formation with the thiol-specific oxidant diamide and chemical cross-linking reactions with the trifunctional maleimide TMEA. CF binding to membranes is mediated by its N-terminal His tag binding to vesicles containing a nickel-chelating lipid, so cross-linking reactions conducted in the presence and absence of vesicles differ only in whether CF is bound to the vesicles or is free in solution. For multiple Cys throughout the CF, membrane association is shown to increase the rate and extent of these reactions. Cross-linking specificity, which is measured as the preference for cross-linking between Cys near each other in the native structure, is also enhanced by membrane association. These results provide an experimental demonstration that membrane binding enhances protein-protein interactions, an important consideration for understanding processes involving membrane-associated proteins. The experiments further demonstrate the importance of cross-linking conditions for these reactions that are often used to probe protein structure and dynamics and the potential of membrane association to restore native interactions of membrane-associated proteins for cross-linking studies.

Two isoforms of Geobacter sulfurreducens PilA have distinct roles in pilus biogenesis, cytochrome localization, extracellular electron transfer, and biofilm formation.

Type IV pili of Geobacter sulfurreducens are composed of PilA monomers and are essential for long-range extracellular electron transfer to insoluble Fe(III) oxides and graphite anodes. A previous analysis of pilA expression indicated that transcription was initiated at two positions, with two predicted ribosome-binding sites and translation start codons, potentially producing two PilA preprotein isoforms. The present study supports the existence of two functional translation start codons for pilA and identifies two isoforms (short and long) of the PilA preprotein. The short PilA isoform is found predominantly in an intracellular fraction. It seems to stabilize the long isoform and to influence the secretion of several outer-surface c-type cytochromes. The long PilA isoform is required for secretion of PilA to the outer cell surface, a process that requires coexpression of pilA with nine downstream genes. The long isoform was determined to be essential for biofilm formation on certain surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxides.

Ligand affinity and kinase activity are independent of bacterial chemotaxis receptor concentration: insight into signaling mechanisms.

Binding of attractant to bacterial chemotaxis receptors initiates a transmembrane signal that inhibits the kinase CheA bound ~300 Å distant at the other end of the receptor. Chemoreceptors form large clusters in many bacterial species, and the extent of clustering has been reported to vary with signaling state. To test whether ligand binding regulates kinase activity by modulating a clustering equilibrium, we measured the effects of two-dimensional receptor concentration on kinase activity in proteoliposomes containing the purified Escherichia coli serine receptor reconstituted into vesicles over a range of lipid:protein molar ratios. The IC(50) of kinase inhibition was unchanged despite a 10-fold change in receptor concentration. Such a change in concentration would have produced a measurable shift in the IC(50) if receptor clustering were involved in kinase regulation, based on a simple model in which the receptor oligomerization and ligand binding equilibria are coupled. These results indicate that the primary signal, ligand control of kinase activity, does not involve a change in receptor oligomerization state. In combination with previous work on cytoplasmic fragments assembled on vesicle surfaces [Besschetnova, T. Y., et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105, 12289-12294], this suggests that binding of ligand to chemotaxis receptors inhibits the kinase by inducing a conformational change that expands the membrane area occupied by the receptor cytoplasmic domain, without changing the number of associated receptors in the signaling complex.

Change of line tension in phase-separated vesicles upon protein binding.

We measured the effect of a model membrane-binding protein on line tension and morphology of phase-separated lipid-bilayer vesicles. We studied giant unilamellar vesicles composed of a cholesterol/dioleoylphosphatidylcholine/palmitoylsphingomyelin mixture and a controlled mole fraction of a Ni-chelating lipid. These vesicles exhibited two coexisting fluid-phase domains at room temperature. Owing to the line tension, σ, between the two phases, the boundary between them was pulled like a purse string so that the smaller domain formed a bud. While observing the vesicles in a microscope, histidine-tagged green fluorescent protein was added, which bound to the Ni-chelating lipid. As protein bound, the vesicle shape changed and the length of the phase boundary increased. The change in morphology was attributed to a reduction of σ between the two phases because of preferential accumulation of histidine-tagged green fluorescent protein-Ni-chelating lipid clusters at the domain boundary. Greater reductions of σ were found in samples with higher concentrations of Ni-chelating lipid; this trend provided an estimate of the binding energy at the boundary, approximately k(B)T. The results show how domain boundaries can lead to an accumulation of membrane-binding proteins at their boundaries and, in turn, how proteins can alter line tension and vesicle morphology.

Inch by inch, row by row.

Bacterial chemotaxis systems have cooperatively interacting clusters of transmembrane receptors and signaling proteins to detect, amplify, integrate and adapt to environmental signals. A recent study provides experimental data to construct a new model of the signaling complex.

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins.

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation--a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration--yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements.

Kinase-active signaling complexes of bacterial chemoreceptors do not contain proposed receptor-receptor contacts observed in crystal structures.

The receptor dimers that mediate bacterial chemotaxis form high-order signaling complexes with CheW and the kinase CheA. From the packing arrangement in two crystal structures of different receptor cytoplasmic fragments, two different models have been proposed for receptor signaling arrays: the trimers-of-dimers and hedgerow models. Here we identified an interdimer distance that differs substantially in the two models, labeled the atoms defining this distance through isotopic enrichment, and measured it with (19)F-(13)C REDOR. This was done in two types of receptor samples: isolated bacterial membranes containing overexpressed, intact receptor and soluble receptor fragments reconstituted into kinase-active signaling complexes. In both cases, the distance found was not compatible with the receptor dimer-dimer contacts observed in the trimers-of-dimers or in the hedgerow models. Comparisons of simulated and observed REDOR dephasing were used to deduce a closest approach distance at this interface, which provides a constraint for the possible arrangements of receptor assemblies.

Comparative genomics of Geobacter chemotaxis genes reveals diverse signaling function.

BACKGROUND: Geobacter species are delta-Proteobacteria and are often the predominant species in a variety of sedimentary environments where Fe(III) reduction is important. Their ability to remediate contaminated environments and produce electricity makes them attractive for further study. Cell motility, biofilm formation, and type IV pili all appear important for the growth of Geobacter in changing environments and for electricity production. Recent studies in other bacteria have demonstrated that signaling pathways homologous to the paradigm established for Escherichia coli chemotaxis can regulate type IV pili-dependent motility, the synthesis of flagella and type IV pili, the production of extracellular matrix material, and biofilm formation. The classification of these pathways by comparative genomics improves the ability to understand how Geobacter thrives in natural environments and better their use in microbial fuel cells. RESULTS: The genomes of G. sulfurreducens, G. metallireducens, and G. uraniireducens contain multiple (approximately 70) homologs of chemotaxis genes arranged in several major clusters (six, seven, and seven, respectively). Unlike the single gene cluster of E. coli, the Geobacter clusters are not all located near the flagellar genes. The probable functions of some Geobacter clusters are assignable by homology to known pathways; others appear to be unique to the Geobacter sp. and contain genes of unknown function. We identified large numbers of methyl-accepting chemotaxis protein (MCP) homologs that have diverse sensing domain architectures and generate a potential for sensing a great variety of environmental signals. We discuss mechanisms for class-specific segregation of the MCPs in the cell membrane, which serve to maintain pathway specificity and diminish crosstalk. Finally, the regulation of gene expression in Geobacter differs from E. coli. The sequences of predicted promoter elements suggest that the alternative sigma factors sigma28 and sigma54 play a role in regulating the Geobacter chemotaxis gene expression. CONCLUSION: The numerous chemoreceptors and chemotaxis-like gene clusters of Geobacter appear to be responsible for a diverse set of signaling functions in addition to chemotaxis, including gene regulation and biofilm formation, through functionally and spatially distinct signaling pathways.

Template-directed self-assembly enhances RTK catalytic domain function.

Receptor tyrosine kinases have become important therapeutic targets because of their involvement in diseases, including cancer. Kinase domains, which are soluble and easily purified, have found widespread use in enzyme inhibitor assays, but these domains do not exhibit full function because they are isolated from the membrane. To address this shortcoming, the authors developed a simple method to restore biologically relevant function by assembling kinase domains on a nanometer-scale template, which imitates the membrane surface. Autophosphorylation of template-assembled tyrosine kinase domains from the insulin, EphB2, and Tie2 receptors led to substantially larger phosphorylation levels compared with domains assayed under conventional conditions. Template-directed assembly increased the total substrate phosphorylation of the insulin and EphB2 receptor kinase domains as much as 60-fold and 15-fold, respectively. In contrast, substrate phosphorylation by template-assembled Tie2 was much lower than conventional conditions. The lower activity observed with the template is more biologically relevant because autophosphorylation of Tie2 is self-inhibitory. These results, as well as the underlying similarity between the organization of template-assembled and natural membrane signaling environments, suggest that template-directed assembly of signaling proteins will provide widespread benefit to basic and applied signal transduction research, especially drug discovery.

Liposome-mediated assembly of receptor signaling complexes.

The reconstitution of membrane-associated protein complexes poses significant experimental challenges. The core signaling complex in the bacterial chemotaxis system is an illustrative example: The soluble cytoplasmic signaling proteins CheW and CheA bind to heterogeneous clusters of transmembrane receptor proteins, resulting in an assembly that exhibits cooperative kinase regulation. An understanding of the basis for the cooperativity inherent in the receptor/CheW/CheA interaction, as well as other membrane phenomena, can benefit from functional studies under defined conditions. To meet this need, a simple method was developed to assemble functional complexes on lipid membranes. The method employs a receptor cytoplasmic domain fragment (CF) with a histidine tag and liposomes that contain a Ni(2+) -chelating lipid. Assemblies of CF, CheW, and CheA form spontaneously in the presence of these liposomes, which exhibit the salient biochemical functions of kinase stimulation, cooperative regulation, and CheR-mediated receptor methylation. Although ligand binding phenomena cannot be studied directly with this approach, other factors that influence kinase stimulation and receptor methylation can be explored systematically, including receptor density and competition among stimulating and inhibiting receptor domains. The template-directed assembly of proteins leads to relatively well-defined samples that are amenable to analysis by a number of methods, including light scattering, electron microscopy, and fluorescence resonance energy transfer. The approach promises to be applicable to many systems involving membrane-associated proteins.

Distributed subunit interactions in CheA contribute to dimer stability: a sedimentation equilibrium study.

The structural domains of the Escherichia coli CheA protein resemble 'beads on a string', since the N-terminal phosphate-accepting (P) domain is joined to the CheY/CheB-binding (B) domain through a flexible linker, and the B domain is in turn joined to the C-terminal dimerization/catalytic/regulatory domains by a second intervening linker. Dimerization occurs primarily via interactions between two dimerization domains, which is required for CheA trans-autophosphorylation. In this study, sedimentation equilibrium was used to demonstrate significant subunit interactions at secondary sites in the two naturally occurring (full-length and short) forms of CheA (CheA(1-654) or CheA(L), and CheA(98-654) or CheA(S)) by contrasting the dimerization of CheA(L) and CheA(S) to CheA(T), an engineered form that lacked the P domain entirely. The estimated dimer dissociation constant (K(1,2)) for CheA(T) (3.1 microM) was weaker than K(1,2) for CheA(L) (0.49 microM), which was attributed to the P domain-catalytic domain interactions that were present in CheA(L) but not CheA(T). In contrast, CheA(S) dimerization was unexpectedly stronger (K(1,2) approximately 20 nM), which arose through interactions between two P domain remnants in the CheA(S) dimer. This conclusion was supported by the results of sedimentation equilibrium experiments conducted with P domains and P domain remnants expressed in the absence of the dimerization/catalytic/regulatory domains. The P domain remnant had a measurable tendency to self-associate; the full-length P domain did not. Hydrophobic forces probably drive this interaction, since hydrophobic amino acids buried in the intact P domain are solvent-exposed in CheA(S). Also, the nascent N-terminus of CheA(S) bound to the phosphatase (CheZ) more effectively, a conclusion based on the demonstrably greater ability of the P domain remnant to co-sediment CheZ, compared to the intact P domain.

Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW.

BACKGROUND: Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr), which contributes to an increase in the steady-state (adapted) methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA) and an adaptor protein (CheW), but their influences on receptor methylation are unknown. Here, the influence of CheW on the rate of Tsr methylation has been studied to identify contributions to the process of adaptation. RESULTS: Methyl group incorporation was measured in a series of membrane samples in which the Tsr molecules were engineered to have one available methyl-accepting glutamate residue (297, 304, 311 or 493). The relative rates at these sites (0.14, 0.05, 0.05 and 1, respectively) differed from those found previously for the aspartate receptor (Tar), which was in part due to sequence differences between Tar and Tsr near site four. The addition of CheW generated unexpectedly large and site-specific rate increases, equal to or larger than the increases produced by serine. The increases produced by serine and CheW (added separately) were the largest at site one, approximately 3 and 6-fold, respectively, and the least at site four, no change and approximately 2-fold, respectively. The rate increases were even larger when serine and CheW were added together, larger than the sums of the increases produced by serine and CheW added separately (except site four). This resulted in substantially larger serine-stimulated increases when CheW was present. Also, CheW enhanced methylation rates when either two or all four sites were available. CONCLUSION: The increase in the rate of receptor methylation upon CheW binding contributes significantly to the ligand specificity and kinetics of sensory adaptation. The synergistic effect of serine and CheW binding to Tsr is attributed to distinct influences on receptor structure; changes in the conformation of the Tsr dimer induced by serine binding improve methylation efficiency, and CheW binding changes the arrangement among Tsr dimers, which increases access to methylation sites.

Mobile loop mutations in an archaeal inositol monophosphatase: modulating three-metal ion assisted catalysis and lithium inhibition.

The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg(2+) for activity and binds three to four ions tightly in the absence of ligands: K(D) = 0.8 muM for one ion with a K(D) of 38 muM for the other Mg(2+) ions. However, the enzyme requires 5-10 mM Mg(2+) for optimum catalysis, suggesting substrate alters the metal ion affinity. In crystal structures of this archaeal IMPase with products, one of the three metal ions is coordinated by only one protein contact, Asp38. The importance of this and three other acidic residues in a mobile loop that approaches the active site was probed with mutational studies. Only D38A exhibited an increased kinetic K(D) for Mg(2+); D26A, E39A, and E41A showed no significant change in the Mg(2+) requirement for optimal activity. D38A also showed an increased K(m), but little effect on k(cat). This behavior is consistent with this side chain coordinating the third metal ion in the substrate complex, but with sufficient flexibility in the loop such that other acidic residues could position the Mg(2+) in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC(50) approximately 250 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li(+) with an IC(50) of 12 mM. These results constitute additional evidence for three metal ion assisted catalysis with substrate and product binding reducing affinity of the third necessary metal ion. They also suggest a specific mode of action for lithium inhibition in the IMPase superfamily.

Template-directed assembly of signaling proteins: a novel drug screening and research tool.

A multitude of proteins reside at or near the cell membrane, which provides a unique environment for organizing and promoting assemblies of proteins that are involved in a variety of cellular signaling functions. Many of these proteins and pathways are implicated in disease. For example, strong links have been established between receptor tyrosine kinases and disease, most notably, cancer. However, a significant impediment to researchers remains: membrane-associated proteins are difficult to reconstitute and study. Template-directed assembly represents a powerful new technology that enables the assembly of membrane-associated proteins. We show that template-directed assembly restores tyrosine kinase activity and regulation, and provides a way for researchers to build multicomponent assemblies. As an example of better enzyme regulation, the Tie2 tyrosine kinase domain exhibits (biologically relevant) autoinhibitory behavior when template assembled. Also, template-assembled insulin receptor tyrosine kinase domains exhibit significant autophosphorylation (none detected without template-directed assembly) and an eightfold increase in substrate phosphorylation (compared to best solution conditions). Thus, template-directed assembly has a demonstrated ability to effectively produce more biologically relevant results using these commercial reagents. Template-directed assembly promises to be generally applicable to the signaling networks important for human health, because these pathways frequently contain membrane-associated proteins that require the organizing influence of a membrane surface.

Formation and activity of template-assembled receptor signaling complexes.

Problems in membrane biology require methods to recreate the interactions between receptors and cytoplasmic signaling proteins at the membrane surface. Here, unilamellar vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and a nickel-chelating lipid were used as templates to direct the assembly of proteins from the Escherichia coli chemotaxis signaling pathway. The bacterial chemoreceptors are known to form clusters, which promote the binding of the adaptor protein (CheW) and the kinase (CheA). When CheA was incubated with vesicles, CheW, and a histidine-tagged cytoplasmic domain fragment of the aspartate chemoreceptor (CF), the kinase activity was stimulated approximately 300-fold. Activity and pull-down assays were used with dynamic light scattering and electron microscopy to characterize the protein-vesicle compositions that were correlated with the high levels of activity, which demonstrated that CF-CheW-CheA complexes on the vesicle surface were the active entities. Assembly and stimulation occurred with vesicles of different sizes and CFs in different extents of glutamine substitution (in place of glutamate) at physiologically relevant sites. An exception was the combination of sonicated vesicles with the unsubstituted CF, which displayed lower CheA activity. The lower activity was attributed to the high curvature of the sonicated vesicles and a weaker tendency of the unsubstituted CF to self-assemble. Electron micrographs of the vesicle-protein assemblies revealed that protein binding induced pronounced changes in vesicle shape, which was consistent with the introduction of positive curvature in the outer leaflet of the bilayer. Overall, vesicle-mediated template-directed assembly is shown to be an effective way to form functional complexes of membrane-associated proteins and suggests that significant changes in membrane shape can be involved in the process of transmembrane signaling.

Competitive and cooperative interactions in receptor signaling complexes.

In bacterial chemotaxis, clustered transmembrane receptors and the adaptor protein CheW regulate the kinase CheA. Receptors outnumber CheA, yet it is poorly understood how interactions among receptors contribute to regulation. To address this problem, receptor clusters were simulated using liposomes decorated with the cytoplasmic domains of receptors, which supported CheA binding and stimulation. Competitive and cooperative interactions were revealed through the use of known receptor signaling mutants, which were used in mixtures with the wild type domain. Competitive effects among the receptor domains sorted cleanly into two categories defined by either stronger or weaker interactions with CheA. Cooperative effects were also evident in CheA binding and activity. In the transition from the stimulating to the inhibiting states, both the cooperativity of the transition and the persistence of stimulation by the wild type domain increased with receptor modification, as in the intact receptor. We conclude that competitive and cooperative receptor interactions both contribute to CheA regulation and that liposome-mediated assembly is effective in addressing these general membrane phenomena.