The nature of the stimulus and of the fumarate binding site of the fumarate sensor DcuS of Escherichia coli

Abstract

DcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C4-dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C4-dicarboxylates without or with substitutions at C2/C3 by hydrophilic (hydroxy, amino, or thiolate) groups stimulated gene expression in a similar way. When one carboxylate was replaced by sulfonate, methoxy, or nitro groups, only the latter (3-nitropropionate) was active. Thus, the ligand of DcuS has to carry two carboxylate or carboxylate/nitro groups 3.1-3.8 Å apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2-3 mM for the C4-dicarboxylates and 0.5 mM for 3-nitropropionate or D-tartrate. The periplasmic domain of DcuS contains a conserved cluster of positively charged or polar amino acid residues (Arg 107-X2-His110-X9-Phe 120-X26-Arg147-X-Phe149) that were essential for fumarate-dependent transcriptional regulation. The presence of fumarate or D-tartrate caused sharpening of peaks or chemical shift changes in HSQC NMR spectra of the isolated C4-dicarboylate binding domain. The amino acid residues responding to fumarate or D-tartrate were in the region comprising residues 89-150 and including the supposed binding site. DcuS(R147A) mutant with an inactivated binding site was isolated and reconstituted in liposomes. The protein showed the same (activation-independent) kinase activity as DcuS, but autophosphorylation of DcuS was no longer stimulated by C 4-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.