Identification of an Anchor Residue for CheA-CheY Interactions in the Chemotaxis System of Escherichia coli

Abstract

Transfer of a phosphoryl group from autophosphorylated CheA (P-CheA) to CheY is an important step in the bacterial chemotaxis signal transduction pathway. This reaction involves CheY (i) binding to the P2 domain of P-CheA and then (ii) acquiring the phosphoryl group from the P1 domain. Crystal structures indicated numerous side chain interactions at the CheY-P2 binding interface. To investigate the individual contributions of the P2 side chains involved in these contacts, we analyzed the effects of eight alanine substitution mutations on CheA-CheY binding interactions. An F214A substitution in P2 caused ϳ1,000-fold reduction in CheA-CheY binding affinity, while Ala substitutions at other P2 positions had small effects (E171A, E178A, and I216A) or no detectable effects (H181A, D202A, D207A, and C213A) on binding affinity. These results are discussed in relation to previous in silico predictions of hot-spot and anchor positions at the CheA-CheY interface. We also investigated the consequences of these mutations for chemotaxis signal transduction in living cells. CheA(F214A) was defective in mediating localization of CheY-YFP to the large clusters of signaling proteins that form at the poles of Escherichia coli cells, while the other CheA variants did not differ from wild-type (wt) CheA (CheA wt ) in this regard. In our set of mutants, only CheA(F214A) exhibited a markedly diminished ability to support chemotaxis in motility agar assays. Surprisingly, however, in FRET assays that monitored receptor-regulated production of phospho-CheY, CheA(F214A) (and each of the other Ala substitution mutants) performed just as well as CheA wt . Overall, our findings indicate that F214 serves as an anchor residue at the CheA-CheY interface and makes an important contribution to the binding energy in vitro and in vivo; however, loss of this contribution does not have a large negative effect on the overall ability of the signaling pathway to modulate P-CheY levels in response to chemoattractants.Chemotaxis in Escherichia coli and numerous other bacterial species involves regulation of the level of phosphorylated CheY (P-CheY) in response to spatial gradients of beneficial and harmful chemicals. P-CheY plays a crucial role in chemotaxis by enabling cells to control how frequently they change directions as they swim (2,48,58,63). The level of P-CheY in a cell reflects the relative rates of phosphorylation (mediated by CheA) and dephosphorylation (mediated by CheZ) (15,46). CheA functions as an autokinase, and this activity is regulated by membrane-spanning receptor proteins responsible for binding chemical ligands that serve as attractants or repellents (7, 16). Autophosphorylated CheA (P-CheA) serves as a phosphodonor for CheY, and the P-CheY generated by this interaction can bind to the switch component of the flagellar motor, inducing changes in cell swimming direction by promoting changes in the direction of flagellar rotation (41,65,66). This sequence of events provides a signal transduction pathway that allows the chemotaxis re...

Robust Signal Processing in Living Cells

“…A further example of a relevant interaction characteristic is the competitive binding of CheY and CheB to CheA, which results in a phosphotransfer rate to CheB that scales as . While not fine-tuned on the parameter level, this qualitative dependence is a prerequisite for robustness of the pathway output and in excellent agreement with experimental findings [21]. In this sense, our approach also offers a theoretical framework to investigate the functional relevance of given reaction characteristics – beyond their role in straightforward signal transmission.…”

regO: a novel locus in the regulation of tetrapyrrole biosynthesis in Rhodospirillum rubrum

“…6). CheY is known as the response regulator of the chemotaxis sensory protein CheA (Thakor et al 2011),(Ann Hirschman, Marina Boukhvalova, Ricaele VanBruggen, Alan J. Wolfe 2001). The predicted cytosolic nature of RegO (see Results) suggests a possible crosstalk between RegO and other response regulators that are likely to obtain the phosphate from RegO.…”

Simultaneously measuring multiple protein interactions and their correlations in a cell by Protein-interactome Footprinting

“…An interaction map of different protein-protein interactions can be discovered by using mutations (CheA (L310S)20, CheA (L126A)21, CheA (F214A)2223, CheZ (L110P)24, CheZ (D143G)24, CheY (D12E)25) to interrupt interactions in this study and measuring global response only. In our study, three interactions are measured simultaneously.…”