Binding of the chemotaxis response regulator CheY to the isolated, intact switch complex of the bacterial flagellar motor: lack of cooperativity.
In bacteria, the chemotactic signal is greatly amplified between the chemotaxis receptors and the flagellar motor. In Escherichia coli, part of this amplification occurs at the flagellar switch. However, it is not known whether the amplification results from cooperativity of CheY binding to the switch or from a post-binding step. To address this question, we purified the intact switch complex (constituting the switch proteins FliG, FliM, and FliN and the scaffolding protein FliF) in quantities sufficient for biochemical work and used it to investigate whether the binding of CheY to the switch complex is cooperative. As a negative control, we used complexes of switchless basal bodies, formed from the proteins FliF and FliG and similarly isolated. Using double-labeling centrifugation assays for binding, we found that CheY binds to the isolated, intact switch complex in a phosphorylation-dependent manner. We observed no significant phosphorylation-dependent binding to the negative control of the switchless basal body. The dissociation constant for the binding between the switch complex and phosphorylated CheY (CheY approximately P) was 4.0 +/- 1.1 microm, well in line with the published range of CheY approximately P concentrations to which the flagellar motor is responsive. Furthermore, the binding was not cooperative (Hill coefficient approximately 1). This lack of CheY approximately P-switch complex binding cooperativity, taken together with earlier in vivo studies suggesting that the dependence of the rotational state of the motor on the fraction of occupied sites at the switch is sigmoidal and very steep (Bren, A., and Eisenbach, M. (2001) J. Mol. Biol. 312, 699-709), indicates that the chemotactic signal is amplified within the switch, subsequent to the CheY approximately P binding.
Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel.
Yael
Y
Sagi
Shahid
S
Khan
Michael
M
Eisenbach
D018407:Bacterial Physiological Phenomena; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005407:Flagella / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C077389:FliN protein, Bacteria; C075661:Flif protein, Bacteria; C075660:Flig protein, Bacteria; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; C066821:FliM protein, Bacteria