A chemotactic signaling surface on CheY defined by suppressors of flagellar switch mutations

Abstract

CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The chemotaxis (Che) proteins couple flagellar rotation to the environment by transducing chemotactic signals from specific transmembrane chemoreceptors to the flagellar switch apparatus. One of these proteins, CheY, is a small (14-kDa), single-domain protein homologous to the regulator proteins of bacterial two-component sensory transduction systems (3) and is the only two-component signaling protein for which the high-resolution crystal structure has been determined (47, 51). Like other regulators, CheY activity appears to be controlled by phosphorylation (reviewed in references 8 and 48); cheY function is required for CW flagellar rotation (34, 35); CheY is phosphorylated in vitro by CheA (16,17,33,54); and cheY or cheA mutations that disrupt phosphotransfer reactions also result in smoothswimming phenotypes, suggesting that CheY-P is the active CW generator (9,33). While CheY is thought to act directly on the switch apparatus to regulate flagellar rotation (11,38,39,53,55), exactly how CheY functions is not known.

Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis

“…Lack of metal ion binding to the mutant protein is consistent with the observation that in wild-type CheY, Asp 13 is directly involved in coordination of the Mg 2ϩ ion (15,16). Furthermore, D13K is phosphorylated only very slowly in the presence of the histidine protein kinase CheA and ATP (26,27), and apparently not at all in the presence of acetyl phosphate (34). Thus, crystallization of CheY D13K in the absence of both Mg 2ϩ and phosphate is biologically relevant.…”

“…Although the lysine side chain at position 13 appears capable of assuming an alternate rotameric position (t) away from the active site with no steric restrictions, its observed position (g Ϫ ) in the structure blocks access to the active site, especially the Mg 2ϩ binding region. This is consistent with the fact that the CheY D13K mutant does not bind Mg 2ϩ (34) and exhibits essentially no phosphorylation ability (26,27,34).…”

“…Genetic evidence that the CheY D13K protein is activated in the absence of phosphorylation implies that the phosphoryl group does not play a direct role in the mechanism of CheY signal transduction, such as phosphotransfer to a downstream protein, or direct binding (25,26). These results, plus biochemical evidence that CheY activation does not involve a change to a multimeric state (25,27), lead to the conclusion that the indirect role of phosphorylation is to generate a conformational change that allows CheY to interact productively with the flagellar switch. NMR data show that CheY undergoes a conformational change upon phosphorylation (28,29).…”

Analysis of a chemotaxis operon from Rhodospirillum centenum

“…While we have no direct evidence for separate motors in R. centenum, we have observed that the polar and lateral flagella are composed of different flagellin and basal ring subunits (16,32). Evidence for different specificities of the R. centenum CheY homologs to the motor complex can also be obtained by inspection of the motor docking domains that have been identified by mutational and crystallographic analyses of CheY homologs from E. coli and S. typhimurium (31,37,38,43). As shown in the CheY alignment in Fig.…”

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

“…3) They had previously been shown to have altered CheA binding (D93K, A90V, Y106W, V108M, F111V, and T112I) (35). 4) They had been identified as suppressors of mutations affecting the flagellar switch (A90V, V108M, F111V, T112I, E117K, and E27K) (9). To test the dephosphorylation rates of mutant CheY proteins, we first needed to know whether the mutant CheY proteins could be phosphorylated by CheA.…”