Isolation and in Vitro Characterization of CheZ Suppressors for the Escherichia coli Chemotactic Response Regulator Mutant CheYN23D

Abstract

The phosphorylated form of the response regulator CheY promotes the tumble signal in Escherichia coli chemotaxis. Phospho-CheY is thought to interact with the switch at the base of the flagellar motor and cause reversal of flagellar rotation from counterclockwise to clockwise changing the swimming direction. Thus the level of phospho-CheY controls the direction of flagellar rotation. The decay of the tumble signal is caused by dephosphorylation of CheY. CheY has an intrinsic autophosphatase activity; however, this reaction is greatly accelerated by the presence of the CheZ protein.We have shown previously that mutations at residues Asn-23 and Lys-26 in CheY confer resistance to the dephosphorylation activity of CheZ (Sanna, M. G., Swanson, R. V., Bourret, R. B., and Simon, M. I. (1995) Mol. Microbiol. 15, 1069 -1079). Here we show that mutant CheY(N23D) is impaired in binding to CheZ, which provides a possible explanation for its resistance to the dephosphorylation activity of CheZ. Moreover, we isolated CheZ second-site suppressors of CheY(N23D), which restore both dephosphorylation and binding activity in a CheY(N23D) background. When the CheZ suppressor mutations are mapped, they are found in two clusters at the N and C termini of the CheZ protein which could define two regions of interaction with CheY. Furthermore, these regions may generate a surface in the folded three-dimensional structure of CheZ required for interaction with CheY.The ability of Escherichia coli to survive in different environments may be the result of its ability to swim toward favorable conditions using the rotation of 6 -8 flagella. This process is called bacterial chemotaxis and has been extensively studied in the past 30 years. When flagella rotate counterclockwise, they promote swimming in one direction for an extensive period of time (smooth swimming); reverse rotation (clockwise) causes tumbling of the cell, thus reorienting the swimming direction. The signal transduction pathway that regulates bacterial chemotaxis consists of the transmembrane receptor proteins Tar, Tsr, Trg, and Tap; the two-component system CheA/CheY; and the CheW and CheZ proteins (for reviews see Refs. 1-4). The external changes detected by the receptor are "communicated" to the histidine kinase CheA, which is able to modulate its autophosphorylation capacity (5, 6). Phosphorylated CheA transfers the phosphate group to the response regulator CheY (7,8), which in the phosphorylated form is able to interact with the flagellar switch and modify the sense of flagellar rotation from counterclockwise to clockwise, promoting tumble motion (9 -13). The CheZ protein accelerates the dephosphorylation of CheY, thus restoring smooth swimming (7,14,15).The mechanism of interaction between CheY and CheZ has not been elucidated, although some observations suggest an allosteric effect of CheZ on CheY instead of canonical phosphatase activity (16). We isolated a mutant CheY(N23D) that is resistant to the dephosphorylating activity of CheZ but is not impaired in phosphorylati...

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

“…1) They contain residue substitutions close to the phosphorylation site of Asp 57 (V86M, T87I, T87S, T87I/Y106W, and A88T). 2) They contain residue substitutions located close to those in previously described CheZ-binding mutants (29,30). 3) They had previously been shown to have altered CheA binding (D93K, A90V, Y106W, V108M, F111V, and T112I) (35).…”

“…2). The residues, altered in these proteins, cluster in the three-dimensional structure of CheY on the same face of CheY as the N23D and K26E CheY mutations that alter CheZ binding (29,30). Fig.…”

“…To explore this possibility, the binding activity of wildtype and mutant CheY proteins with CheZ was measured. CheZ binds to the phosphorylated form of CheY with higher affinity than it does to the unphosphorylated form of CheY (24,26,30). Therefore, binding of apo-CheY and CheY-P to CheZ proteins was measured.…”

“…The carboxyl-terminal domain in CheZ has been identified as the CheY-binding domain (27). The CheZ-binding region of CheY has not been completely elucidated, although two mutant CheY proteins with reduced ability to bind CheZ have been reported (29,30). In this study, we characterize a number of mutant CheY proteins as substrates for CheZ.…”

“…The binding of CheZ to CheY-P is greater than its binding to apo-CheY (24,30). The carboxyl-terminal domain in CheZ has been identified as the CheY-binding domain (27).…”