Investigation of the role of electrostatic charge in activation of the Escherichia coli response regulator CheY.

Journal Article (Journal Article)

In a two-component regulatory system, an important means of signal transduction in microorganisms, a sensor kinase phosphorylates a response regulator protein on an aspartyl residue, resulting in activation. The active site of the response regulator is highly charged (containing a lysine, the phosphorylatable aspartate, two additional aspartates involved in metal binding, and an Mg(2+) ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties. Furthermore, substitution of one of the Mg(2+)-coordinating aspartates with lysine or arginine in the Escherichia coli chemotaxis response regulator CheY results in phosphorylation-independent activation. In order to examine the consequences of altered charge distribution for response regulator activity and to identify possible additional amino acid substitutions that result in phosphorylation-independent activation, we made 61 CheY mutants in which residues close to the site of phosphorylation (Asp57) were replaced by various charged amino acids. Most substitutions (47 of 61) resulted in the complete loss of CheY activity, as measured by the inability to support clockwise flagellar rotation. However, 10 substitutions, all introducing a new positive charge, resulted in the loss of chemotaxis but in the retention of some clockwise flagellar rotation. Of the mutants in this set, only the previously identified CheY13DK and CheY13DR mutants displayed clockwise activity in the absence of the CheA sensor kinase. The absence of negatively charged substitution mutants with residual activity suggests that the introduction of additional negative charges into the active site is particularly deleterious for CheY function. Finally, the spatial distribution of positions at which amino acid substitutions are functionally tolerated or not tolerated is consistent with the presently accepted mechanism of response regulator activation and further suggests a possible role for Met17 in signal transduction by CheY.

Full Text

Duke Authors

Cited Authors

  • Smith, JG; Latiolais, JA; Guanga, GP; Citineni, S; Silversmith, RE; Bourret, RB

Published Date

  • November 2003

Published In

Volume / Issue

  • 185 / 21

Start / End Page

  • 6385 - 6391

PubMed ID

  • 14563873

Pubmed Central ID

  • PMC219398

International Standard Serial Number (ISSN)

  • 0021-9193

Digital Object Identifier (DOI)

  • 10.1128/JB.185.21.6385-6391.2003


  • eng

Conference Location

  • United States