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

Abstract

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.

Fig. 1.

The chemoreceptor dimer and membrane-assembled signaling complexes. (A) The receptor dimer (green) depicted with thickened line segments for α-helices and domains labeled (Ligand Binding, HAMP, Cytoplasmic). W (magenta) and the A dimer (A₂, red) are bound to the c-domain, and the four major methylation sites are shown partly modified (QEQE) as filled (sites 1 and 3 modified) and unfilled (unmodified) circles. (B) QEQE c-domain dimers (CF, green) are assembled with A₂ and W on a vesicle outer surface (blue). (C) A membrane section viewed from above, in which CF dimers (green circles) are shown (in cross-section) bound end-on to the lipids (blue capsules), corresponding to a situation with 30 μM CF and 580 μM total lipid. The CF cross-sectional area (≈4 nm²) is scaled to depict the surface area occupied by CF at a lipid/CF ratio of 20 (≈14 nm² per CF dimer).

Fig. 2.

Kinase activity versus CF surface concentration. (A) Kinase activity of CheA assembled on SUVs with CheW and CF_4E, CF_QEQE, or CF_4Q (open, gray, and black, respectively) containing nickel lipid at the stated mole percentages. (B) Activity of CheA assembled on LUVs (as in A). (C) Cooperative increase in the activity of CheA assembled, with W and CF_4E, on membrane vesicles containing the stated mole percentages of nickel lipid: SUVs (open circles) and LUVs prepared by extrusion through filters with pore diameters of 50 nm (filled circles) or 100 nm (open squares). The lines are fits of the data to the Hill equation, as described in SI Materials and Methods. (D) f_B(CheA), the fraction of CheA that co-sedimented with CF-decorated vesicles (symbols as in C). Protein and lipid concentrations: 30 μM CF, 5.0 μM W, 1.2 μM CheA, 290 μM nickel-chelating lipid, and DOPC (to achieve the stated mole percentage of nickel lipid). Activities and uncertainties are means and standard deviations of triplicates, respectively.

Fig. 3.

FRET efficiency, kinase activity, and methylation rate versus CF surface concentration. (A) Kinase activity (open symbols) and FRET efficiency (filled symbols) of membrane-assembled A, W, and donor- and acceptor-labeled mixtures of CF_4E (circles) or CF_4Q (squares and triangles). The kinase activities shown were measured with donor-labeled/unlabeled CF mixtures. Acceptor-labeled/donor-labeled/unlabeled CF mixtures produced similar results, although the activities measured with labeled CFs were generally smaller than activities observed with unlabeled CFs. Activities and uncertainties are means and standard deviations of triplicates, respectively. LUVs were made with 50-nm pore-diameter filters. Concentrations at assembly: 30 μM CF, 1.2 μM A, 5 μM W, 290 μM nickel-chelating lipid, and DOPC (to achieve the stated mole percentage of nickel lipid). (B) Methylation (pmol of CH₃/min) of template-assembled CF_4E versus surface concentration, without A and W (open bars) and with 5.0 μM W and 1.2 μM A (filled bars).

Fig. 4.

Effects of surface concentration on kinase activity and methylation combine to regulate signaling. (A) Kinase activities (circles) and methylation rates (bars) versus surface concentration (CF per nm²) of CF_4E–A–W complexes (from Figs. 2C and 3B, respectively). (B) A model based on low- and high-density states and the factors that influence their formation (curved arrows). Arrays receptor dimer (green), W (magenta), and A (red) reside on the membrane (blue lines) in low- or high-density states (left and right, respectively). At high density, the methylation level, represented by filled circles, is larger, and CheA is stimulated to phosphorylate CheY (Y, yellow) and the methylesterase CheB (B, brown). Formation of the low-density state is promoted by attractant binding (filled circles) and receptor demethylation by phospho-CheB (CheB-P). In the low-density state, receptors do not stimulate CheA, and attractant-bound receptors are methylated more rapidly by CheR (R, blue), which favors formation of the high-density state.