No abstract

Sensor histidine kinases of two-component signal-transduction systems are essential for bacteria to adapt to variable environmental conditions. However, despite their prevalence, it is not well understood how extracellular signals such as ligand binding regulate the activity of these sensor kinases. CitA is the sensor histidine kinase in Klebsiella pneumoniae that regulates the transport and anaerobic metabolism of citrate in response to its extracellular concentration. We report here the X-ray structures of the periplasmic sensor domain of CitA in the citrate-free and citrate-bound states. A comparison of the two structures shows that ligand binding causes a considerable contraction of the sensor domain. This contraction may represent the molecular switch that activates transmembrane signaling in the receptor.

SummaryThe two-component regulatory system CitA /CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain¯anked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE±CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327±547) showed constitutive autokinase activity and transferred the g-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB±D56N formed a stable complex with MalE±CitAC.

Enzyme I (EI) is the phosphoenolpyruvate (PEP)-protein phosphotransferase at the entry point of the PEP-dependent sugar phosphotransferase system, which catalyzes carbohydrate uptake into bacterial cells. In the first step of this pathway EI phosphorylates the heat-stable phospho carrier protein at His-15 using PEP as a phosphoryl donor in a reaction that requires EI dimerization and autophosphorylation at His-190. The structure of the full-length protein from Staphylococcus carnosus at 2.5 Å reveals an extensive interaction surface between two molecules in adjacent asymmetric units. Structural comparison with related domains indicates that this surface represents the biochemically relevant contact area of dimeric EI. Each monomer has an extended configuration with the phosphohistidine and heat-stable phospho carrier protein-binding domains clearly separated from the C-terminal dimerization and PEP-binding region. The large distance of more than 35 Å between the active site His-190 and the PEP binding site suggests that large conformational changes must occur during the process of autophosphorylation, as has been proposed for the structurally related enzyme pyruvate phosphate dikinase. Our structure for the first time offers a framework to analyze a large amount of research in the context of the full-length model.Group translocation is the membrane transport mechanism by which a substrate is chemically modified to an impermeable derivative as it crosses the cell membrane. This energy-efficient transport strategy is used by bacteria for the uptake of rapidly metabolizable sugars, and it is achieved through a highly conserved three component phospho-relay system called the phosphoenolpyruvate:sugar phosphotransferase system (PTS) 4 (1-3). The PTS catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to a sugar while it is being transported across the membrane. It consists of two universal components, Enzyme I (molecular mass, 63 kDa) (4, 5), hereafter referred to as EI, and the heat-stable histidine phospho carrier protein (HPr) (molecular mass, 9 kDa), and in addition several membrane-associated components, which are sugar-specific and are collectively designated as Enzyme II (EII) complexes (3). The PTS cascade starts with the autophosphorylation of EI on a conserved histidine (His-190 in the Staphylococcus carnosus EI studied in this report) in a reaction that uses PEP as phosphoryl donor (4). Subsequently, the phosphoryl group is transferred to His-15 of the HPr protein, and ultimately to the imported hexose, in a series of transphosphorylation reactions mediated by the components of the sugar-specific EII complex. The PTS is not only responsible for sugar uptake; it also represents a major sensing and signaling system in the bacterial cell. The phosphorylation state of the components of the PTS pathway is directly coupled to the regulation of carbohydrate metabolism, chemotaxis toward carbon sources (6), carbon catabolite repression (7,8), and nitrogen metabolism (9). Because EI catalyze...

The sensor kinase CitA and the response regulator CitB of Klebsiella pneumoniae form the paradigm of a subfamily of bacterial two-component regulatory systems that are capable of sensing tri- or dicarboxylates in the environment and then induce transporters for the uptake of these compounds. We recently showed that the separated periplasmic domain of CitA, termed CitAP (encompasses residues 45-176 supplemented with an N-terminal methionine residue and a C-terminal hexahistidine tag), is a highly specific citrate receptor with a K(d) of 5.5 microM at pH 7. To identify positively charged residues involved in binding the citrate anion, each of the arginine, lysine, and histidine residues in CitAP was exchanged for alanine, and the resulting 17 muteins were analyzed by isothermal titration calorimetry (ITC). In 12 cases, the K(d) for citrate was identical to that of wild-type CitAP or slightly changed (3.9-17.2 microM). In one case (R98A), the K(d) was 6-fold decreased (0.8 microM), whereas in four cases (R66A, H69A, R107A, and K109A) the K(d) was 38- to >300-fold increased (0.2 to >1 mM). The secondary structure of the latter five proteins in their apo-form as deduced from far-UV circular dichroism (CD) spectra did not differ from the apo-form of wild-type CitAP; however, all of them showed an increased thermostability. Citrate increased the melting point (T(m)) of wild-type CitAP and mutein R98A by 6.2 and 9.5 degrees C, respectively, but had no effect on the T(m) of the four proteins with disturbed binding. Three of the residues important for citrate binding (R66, H69, and R107) are highly conserved in the CitA subfamily of sensor kinases, indicating that they might be involved in ligand binding by many of these sensor kinases.