Abstract
Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.
Highlights
Bacterial cells possess phosphotransfer signaling mechanisms known as ‘two-component regulatory systems’ that elicit a variety of adaptive responses to the cells’ environments [1, 2]
A phosphoryl group moves from ATP to the histidine kinase (HK) domain (His residue); secondly, it moves to the receiver domain (Asp residue); and it moves to the histidine-containing phosphotransmitter (HPt) domain (His residue)
We introduce Phos-tag SDS-PAGE as a simple method for identifying whether the reactions of autophosphorylation and phosphoryl transfer in tripartite sensor kinases (TSKs) occur in a cis or in a trans mode
Summary
Bacterial cells possess phosphotransfer signaling mechanisms known as ‘two-component regulatory systems’ that elicit a variety of adaptive responses to the cells’ environments [1, 2] Each of these systems generally consists of a histidine sensor kinase and a response regulator. Some histidine sensor kinases, known as tripartite sensor kinases (TSKs), have a more complex type of phosphorelay consisting of two additional domains: a receiver domain containing a conserved Asp residue, and a histidine-containing phosphotransmitter (HPt) domain. In such systems, signals are transmitted through a more sophisticated threestep phosphorelay. The HPt-phosphorylated TSKs phosphorylate the receiver domain (Asp residue) of response regulators
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