RESUMO
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.
Assuntos
Quimiotaxia , Complexos Multiproteicos , Receptores de Superfície Celular , Transdução de Sinais , Proteínas de Bactérias , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana , Proteínas Quimiotáticas Aceptoras de Metil , Metilação , Proteínas QuinasesRESUMO
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.
Assuntos
Proteínas de Bactérias/fisiologia , Bioquímica/métodos , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Lipossomos/química , Proteínas de Membrana/fisiologia , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Escherichia coli/química , Histidina/química , Histidina Quinase , Lipídeos/química , Proteínas de Membrana/química , Proteínas Quimiotáticas Aceptoras de Metil , Metilação , Modelos Biológicos , Modelos Químicos , Níquel/química , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
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.