Abstract
CheY is a response regulator in the well studied two-component system that mediates bacterial chemotaxis. Phosphorylation of CheY at Asp(57) enhances its interaction with the flagellar motor. Asn(59) is located near the phosphorylation site, and possible roles this residue may play in CheY function were explored by mutagenesis. Cells containing CheY59NR or CheY59NH exhibited hyperactive phenotypes (clockwise flagellar rotation), and CheY59NR was characterized biochemically. A continuous enzyme-linked spectroscopic assay that monitors P(i) concentration was the primary method for kinetic analysis of phosphorylation and dephosphorylation. CheY59NR autodephosphorylated at the same rate as wild-type CheY and phosphorylated similarly to wild type with acetyl phosphate and faster (4-14x) with phosphoramidate and monophosphoimidazole. CheY59NR was extremely resistant to CheZ, requiring at least 250 times more CheZ than wild-type CheY to achieve the same dephosphorylation rate enhancement, whereas CheY59NA was CheZ-sensitive. However, several independent approaches demonstrated that CheY59NR bound tightly to CheZ. A submicromolar K(d) for CheZ binding to CheY59NR-P or CheY.BeF(3)(-) was inferred from fluorescence anisotropy measurements of fluoresceinated-CheZ. A complex between CheY59NR-P and CheZ was isolated by analytical gel filtration, and the elution position from the column was indistinguishable from that of the CheZ dimer. Therefore, we were not able to detect large CheY-P.CheZ complexes that have been inferred using other methods. Possible structural explanations for the specific inhibition of CheZ activity as a result of the arginyl substitution at CheY position 59 are discussed.
Highlights
Flagellated bacteria such as Escherichia coli and Salmonella typhimurium move toward chemical attractants and away from repellants by regulating the frequency with which their flagella switch between counterclockwise (CCW)1 rotation
CheY59NR was extremely resistant to CheZ, requiring at least 250 times more CheZ than wild-type CheY to achieve the same dephosphorylation rate enhancement, whereas CheY59NA was CheZ-sensitive
It is plausible that nonconserved residues in the vicinity of the active sites of response regulators have evolved to regulate the rates of the phosphorylation reactions in a way that meets the individual needs of a system
Summary
Chemicals—The calcium salt of monophosphoimidazole (MPI) [23] and the potassium salt of phosphoramidate (PAM) [24] were synthesized as described. Kinetics of Phosphate Release—The steady state rates of release of inorganic phosphate from reactions containing CheY, MPI, and CheZ were measured using an enzyme-linked spectroscopic assay (Enzchek Pi Kit, Molecular Probes). In this assay, the reactions of interest were carried out in the presence of purine nucleoside phosphorylase and a guanine analog substrate, 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG). Acetyl phosphate (final concentration of 20 mM) was added to the CheY/CheZ mixtures, allowed to incubate for 3 min to allow for maximal phosphorylation, and chromatographed on the column that had been equilibrated with freshly prepared 50 mM Tris, pH 7.5, 10 mM MgCl2, 20 mM acetyl phosphate immediately before application of the sample. Molecular weight standards (Bio-Rad gel filtration standards kit and other individual proteins from Sigma) were independently chromatographed on the same column for estimation of the molecular weight of the species eluting from the column
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