Abstract
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.
Highlights
The clusters of chemoreceptors are heterogeneous with respect to the specificity and mode of ligand binding [5]
These complexes had been assembled on vesicles to demonstrate that wild type CF (WT-CF) stimulated CheA at all levels of adaptational modification [26]
The specific activities of the CheA bound to vesicles decorated with WT-CF, NS-CF, or a 1:1 WT-CF/NS-CF mixture are plotted in Fig. 2, A–D, and E–H shows the fraction of CheA bound to these vesicles (fB (CheA)), and I–L shows the fraction of bound CheW (fB (CheW))
Summary
Proteins and Protein Purification—Histidine-tagged CFs of the aspartate receptor were prepared from the expression plasmids pSM100, pHTCF, and pSM101, which produced CF in low (CF4E), intermediate (CFQEQE), and high (CF4Q) levels of glutamine modification to mimic receptor methylation [26]. These experiments used vesicles with a 1.5 to 1 molar ratio of Ni2ϩ-DOGS to DOPC at a total lipid concentration of 500 and 30 M CF. Where Acto and Act* are the CheA activities bound to 100% WT-CF and 100% NS-CF arrays, respectively; and [CoA] and [C*A] are the CheA concentrations bound to WT- and NS-CFs, respectively (in the presence of CheW), estimated through the model with constraints imposed by the activity and binding data. Cooperative changes in CheA activity and the fraction of bound CheA, fB, were fit to Equation 3, Xn Act ϭ Acto Ϫ ⌬Act X1n/2 ϩ Xn (Eq 3). Cooperative changes in fB, except that Act, Acto, and ⌬Act were replaced by the experimental values of fB, an estimate for fB with vesicles of 100% WT-CF ( f Bo), and the difference in fB between 100% WT-CF and 100% NS-CF (⌬fB)
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