Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria.

Abstract

Regulation of the direction of flagellar rotation is central to the mechanism of bacterial chemotaxis. The transitions between counterclockwise and clockwise rotation are controlled by a "switch complex" composed of three proteins (FUG, FHlM, and FUN) CheY (8,9). In this way, the flux of information from the receptors is integrated into a common form: phosphorylated CheY. Changes in the phosphorylation level ofCheY are believed to be sensed by a group ofproteins located on the cytoplasmic face of the flagellar motor known as the "switch complex" (10), which causes the motor to adopt either a clockwise bias (if levels of phosphorylated CheY are high) or a counterclockwise bias (when phosphorylated CheY is low) (11). The mechanism of switch function is not known. Overproduction of CheY in a strain lacking all other chemotaxis proteins (12-14) or insertion of purified CheY into cell envelopes devoid of cytoplasm (15) MATERIALS AND METHODSPreparation of CheY Beads. CheY beads and control beads were prepared in parallel under identical conditions. Two samples (1-g dry weight each) of CNBr-activated Sepharose 4B beads (Pharmacia) were washed five times in 14 ml of 1 mM HCl and then three times in coupling buffer (0.1 M NaHC03/0.5 M NaCl, pH 8.3). CheY [5-45 mg, determined from absorbance at 280 nm by using a molar extinction coefficient of 6970 M-1"cm-1 (19)] purified from Escherichia coli (11) was dialyzed twice against 2 liters of coupling buffer and added to one tube. An equal volume of coupling buffer was added to the other tube (containing control beads). Both tubes were mixed end-over-end overnight at 4°C. In this way 95-99% ofthe CheY became covalently immobilized. Bovine serum albumin (BSA, 20 mg), previously dialyzed against coupling buffer, was then added to both tubes in order to block unreacted cyano groups, and the incubation with mixing continued for 5-10 hr at 4°C. To complete the coupling, additional BSA (20 mg), this time in 0.1 M Tris HCl (pH 7.9), was added to both tubes and incubated further for 5-10 hr. Finally the beads were washed in 50 mM Tris-HCl (pH 7.9) and stored at 4°C. Prior to use in the binding assay, the beads were washed and resuspended in 8-12 ml of Tris buffer. CheY57DE was produced and isolated as described earlier (20)

Characterization of the chemotaxis protein CheW from Rhodobacter sphaeroides and its effect on the behaviour of Escherichia coli

“…The phosphorylation of CheY at a conserved aspartate residue (D57 ) activates CheY (Bourret et al, 1990). Interaction of the phosphorylated CheY protein with the FliM of the motor switch complex stimulates clockwise motor rotation (Welch et al, 1993). The signal is terminated by CheZ, which stimulates the autophosphatase activity of CheY-phosphate.…”

The CheZ-binding Surface of CheY Overlaps the CheA- and FliM-binding Surfaces

“…In contrast, CheZ and FliM bind to CheY-P with a higher affinity than to apo-CheY (10,11,24,30). It is possible that the overlap region in the unphosphorylated state contributes to the CheA-binding surface (35) and that CheY phosphorylation alters the topology of this region, causing the release of CheY from CheA and increasing the affinity 3 D. Shukla, X. Zhu, and P. Matsumura, submitted for publication.…”

“…The CheZ-binding Surface Partially Overlaps the FliM-and CheA-binding Surfaces of CheY-CheY is a single-domain protein that interacts with at least three other polypeptides: CheA (35,42,43), CheZ (24,26,30), and FliM (10,11,36). Previous studies (35) showed that the mutant CheY proteins A90V, E93K, Y106W, V108M, F111V, T112I, and E117K have altered CheA binding (Table III).…”

Chemotaxis inCampylobacter Jejuni

“…Non-phosphorylated CheY interacts weakly with the FliM switch proteins of the flagellar motor, which are the final effectors of the sensory transduction chain [42][43][44][45]. Because of the only weak interaction between unphosphorylated CheY and FliM, CheY is not able to break up the FliNFliN interaction within the FliN-dimer via the N-terminal hydrophobic patch [46].…”