Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors.

Abstract

Bacterial motility and gene expression are controlled by a family of phosphorylated response regulators whose activities are modulated by an associated family of protein-histidine kinases. In chemotaxis there are two response regulators, CheY and CheB, that' a residue that is conserved in all homologous response regulator proteins (5). Two additional highly conserved residues, Asp-12 and Asp-13, bind a Mg2+ ion that is essential for phosphorylation (6, 7). CheY, like many other response regulators, has an associated autophosphatase activity; phospho-CheY has a half-life of =10 sec. Phosphatase activity is enhanced by an auxiliary regulatory protein, CheZ (8).The activity of response regulators is controlled by a family of histidine kinases that are autophosphorylated in the presence of ATP (1-3). The phosphotransfer mechanism involves the intermediate formation of a phosphohistidine residue in the kinase. In chemotaxis, the rate of phosphorylation of the histidine residue in the kinase, CheA, is stimulated by membrane chemoreceptor proteins (9, 10). The phosphoryl group is then rapidly transferred to CheY to control motility. Another chemotaxis response regulator, CheB, also accepts phosphoryl groups from CheA. -CheB provides a feedback adaptation mechanism. Phosphorylation of its N-terminal regulatory domain stimulates a C-terminal catalytic activity that modifies the chemoreceptors to attenuate CheA kinase activity (9, 11).Although in many cases a specific kinase has been implicated in the regulation of a given response regulator, considerable cross specificity has also been observed (12-14). The phenotypes of kinase mutants have indicated that response regulators can be phosphorylated in the absence of their cognate kinases (15-20). Here we show that CheY and CheB are enzymes that catalyze their own phosphorylation using low molecular weight phospho-donors. Thus, the enzymology of aspartate phosphorylation is an inherent property of the response regulators that can occur independently of any other protein. Proteins. Wild-type and mutant CheY proteins as well as CheZ and CheB were purified as described (23-25). Protein purity was estimated to be >95% on the basis of SDS/PAGE. MATERIALS AND METHODS Materials[3H]Methyl-labeled Tar receptor in Escherichia coli membranes was prepared as described (25 718The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP

“…In order to obtain specific binding of the NarL protein to the DNA, it was necessary to incubate it with acetyl phosphate prior to the binding experiments, indicating that phosphorylation of the protein is a prerequisite for specificity (14,16). Binding constants ranged from 1.2 to 1.7 M, which was of the same order of magnitude as the range found for the constitutive mutant form of the protein NarL (V88A) used in a previous study (14).…”

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

“…It has been suggested that this reaction is catalyzed by CheY rather than CheA (45,46). The claim that CheY possesses kinase activity is supported by the observation that small molecule phosphodonors such as acetyl phosphate can act in place of CheA-P to donate the phosphoryl group to CheY (47,48). The T87I and T87I/Y106W mutant CheY proteins, which have a structural shift in their 90's loop, cause severe defects in autodephosphorylation and then are completely resistant to CheZ activity (Table II).…”

Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis

“…Mg 2ϩ is required in the phosphorylation and dephosphorylation reactions of wild-type CheY (5,21,33). However, Mg 2ϩ binding to CheY D13K is not detectable by either resonance shifts in 19 F NMR spectra of 4-fluorophenylalanine-labeled protein or by quenching of Trp 58 fluorescence (28,34).…”

“…The second component is a response regulator protein, which transfers the phosphoryl group from the histidine protein kinase to itself. Response regulators are also capable of autophosphorylation using small molecule phosphodonors such as acetyl phosphate (5,6). Phosphorylation alters the activity of the response regulator, thereby generating a response (e.g.…”