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J Biol Chem. 2010 Jan 15;285(3):1870-8. doi: 10.1074/jbc.M109.072108. Epub 2009 Oct 28.

A PAS domain binds asparagine in the chemotaxis receptor McpB in Bacillus subtilis.

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1
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

Abstract

During chemotaxis toward asparagine by Bacillus subtilis, the ligand is thought to bind to the chemoreceptor McpB on the exterior of the cell and induce a conformational change. This change affects the degree of phosphorylation of the CheA kinase bound to the cytoplasmic region of the receptor. Until recently, the sensing domains of the B. subtilis receptors were thought to be structurally similar to the well studied Escherichia coli four-helical bundle. However, sequence analysis has shown the sensing domains of receptors from these two organisms to be vastly different. Homology modeling of the sensing domain of the B. subtilis asparagine receptor McpB revealed two tandem PAS domains. McpB mutants having alanine substitutions in key arginine and tyrosine residues of the upper PAS domain but not in any residues of the lower PAS domain exhibited a chemotactic defect in both swarm plates and capillary assays. Thus, binding does not appear to occur across any dimeric surface but within a monomer. A modified capillary assay designed to determine the concentration of attractant where chemotaxis is most sensitive showed that when Arg-111, Tyr-121, or Tyr-133 is mutated to an alanine, much more asparagine is required to obtain an active chemoreceptor. Isothermal titration calorimetry experiments on the purified sensing domain showed a K(D) to asparagine of 14 mum, with the three mutations leading to less efficient binding. Taken together, these results reveal not only a novel chemoreceptor sensing domain architecture but also, possibly, a different mechanism for chemoreceptor activation.

PMID:
19864420
PMCID:
PMC2804345
DOI:
10.1074/jbc.M109.072108
[Indexed for MEDLINE]
Free PMC Article
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