The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di-and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The fumarate sensor DcuS is a prototype for a two component sensory histidine kinase with signal perception in the periplasm, transmembrane signal transfer (1, 2), and autophosphorylation of a His residue in the kinase domain in the cytoplasm (3). DcuS belongs to the CitA family of sensors that are specific for sensing di-and tricarboxylates (1, 2, 4, 5). The periplasmic domain of the histidine autokinase CitA works as a highly specific citrate receptor, whereas DcuS uses any type of C 4 -dicarboxylate, like fumarate, succinate, and malate, as a stimulus (1, 4 -6). DcuS is predicted to consist of two transmenbrane helices and of a periplasmic sensory domain enclosed by the transmembrane helices. The second transmembrane helix is followed by a cytoplasmic PAS 1 domain of unknown function and the kinase with the consensus histidine residue for autophosphorylation. The periplasmic citrate binding domain of CitA is conserved in DcuS and presumably responsible for binding of fumarate and other C 4 -dicarboxylates. Preliminary results suggest that fumarate sensing occurs by this domain in the periplasm (2, 4, 5). After phosphorylation by DcuS the response regulator DcuR of the DcuSR system activates the expression of the target genes like dcuB and frd-ABCD encoding an anaerobic fumarate carrier DcuB and fumarate reductase (4, 5). Despite their prevalence no structural information is available for transmembranous sensory kinases, in particular not for signal perception and transmission across the membrane. Only the structures of cytoplasmic sensory kinases, or of domains not involved in transmembrane signaling, have been determined.Purified DcuS is active after reconstitution in proteoliposomes and capable of transmembranous stimulation of the kinase by fumarate (2). For a more detailed understanding of signal perception representing the first step of signal transduction in transmembranous histidine kinases of two-component systems, the structure of the periplasmic C 4 -dicarboxylate binding domain of DcuS (DcuS-pd) was determined after stable over-production of the domain. ("DcuS-pd")-The sequence of dcuS coding for the periplasmic domain of DcuS (DcuS or DcuS-pd) enclosed by the two transmembrane helices was cloned into the NdeI and HindIII sites of plasmid pET28a (Novagen) resulting in plasmid pMW145. The DNA fragment was amplified with oligonucleotides pdcus-NdeII (ATT TAC TTC TCG CAT ATG AGT GAT ATG) and pdcuSHind (GAC CAG ATA AAG CTT CAG CGA CTG) by PCR of genomic Escheric...
