Bacterial cells possess phosphotransfer signaling mechanisms known as ‘two-component regulatory systems’ that elicit a variety of adaptive responses to the cells’ environments.Each
of these systems generally consists of a histidine sensor kinase and a response regulator.The
sensor kinase senses extra- and intracellular stimuli and regulates the function of its cognate
response regulator through a phosphorylation reaction. Accordingly, the response regulator mediates certain changes in gene expression or cell behavior.
A typical sensor kinase has a histidine kinase (HK) domain containing an invariant His residue that is autophosphorylated in an ATP-dependent manner, whereas a typical response regulator has a receiver domain containing a conserved Asp residue that can acquire a phosphoryl group from its cognate sensor kinase. Most two-component systems have this type of a simple His–Asp phosphorelay scheme.
However, some histidine sensor kinases, including ArcB, 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. First, a phosphoryl group moves from ATP to the HK domain (His residue); secondly, it moves to the receiver domain (Asp residue); and finally it moves to the HPt domain (His residue). Subsequently, the HPt-phosphorylated TSKs phosphorylate the receiver domain (Asp residue) of response regulators.
A typical sensor kinase demonstrated that phosphate group of ATP bound to one monomer subunit transferd to the His residue in the other subunit in the homodimer; this confirmed
that the autophosphorylation reaction of E. coli EnvZ occurs in an intermolecular (trans)
The mode of the three-step phosphorelay from ATP to the terminal HPt domain in TSKs
remains unclear in most cases. In this study, we introduce Phos-tag SDS-PAGE as a simple method for identifying whether the reactions of autophosphorylation and phosphoryl transfer in TSKs occur in a cis or in a trans mode.
We performed a complementation assay between the ArcB G2(ATP binding site was mutated) and H292A (pHis site was mutated) mutants to confirm the intramolecular nature of the autophosphorylation of ArcB. It has been demonstrated that heterodimers of ArcB are also formed by mixing the both mutants under the conditions for the in vitro complementation assay.The reaction products were analyzed by Phos-tag SDS-PAGE.
The WT protein was successfully autophosphorylated, and Phos-tag SDS-PAGE permitted us to detect an upshifted band corresponding to the autophosphorylated ArcB form H292–P containing a phosphorylated H292 residue (indicated by the arrow on the right-hand side of Fig). On the other hand, no upshifted band was detected in the G2 or H292A mutants, showing that these mutants do not autophosphorylate. In the autophosphorylation reaction using a mixed sample of G2 and H292A mutants in equal proportions, no upshifted band was detected, once more showing that complementary autophosphorylation between the two mutants did not occur.
The complementation assay therefore demonstrated that ArcB autophosphorylates in a cis
To determine the mode of the subsequent multistep phosphorelay, we performed three
additional complementation assays between the ArcB H292A and D576A mutants, the H292A
and H717A mutants, and the D576A and H717A mutants, respectively, in the presence of
ArcA. We observed a single upshifted band corresponding to the phosphorylated ArcA (ArcA–P) in the phosphorelay reaction with the ArcB WT, showing that a phosphoryl-transfer reaction from the ArcB WT to ArcA had occurred.
For the phosphorelay reaction with the H292A mutant, no upshifted band of ArcB was observed, whereas a single upshifted band corresponding to the phosphorylated form H292–P was detected in the phosphorelay reaction with the D576A mutant, and two upshifted bands corresponding to the phosphorylated forms H292–P and D576–P were detected in the phosphorelay reaction with the H717A mutant. In the phosphorelay reaction using an equal mixture of H292A and D576A mutants in the presence of ArcA, an upshifted band corresponding to the autophosphorylated ArcB of H292–P was observed, and the phosphoryl group was transferred to ArcA. This result demonstrated that exchange of subunits between the two dimeric ArcB mutants (H292A and D576A) readily occurs and that the complementary phosphotransfer reaction from the H292 residue to the D576 residue occurs intermolecularly (the trans mode) [(ii)], indicating that the dimeric ArcB protein, like the EnvZ protein, is thermodynamically stable but kinetically labile.
In the phosphorelay reactionusing an equal mixture of H292A and H717A mutants, on the other hand, although two upshifted bands corresponding to the phosphorylated ArcB of H292–P and D576–P were observed, no upshifted band of ArcA was detectable. Furthermore, in the phosphorelay reaction using an equal mixture of D576A and H717A mutants, we detected a single upshifted band of ArcA. These results indicate that the phosphoryl-transfer reaction from the D576 residue to the H717 residue proceeds through a trans mode [(iii) and (iv)].
We therefore concluded that the primary ArcB autophosphorylation reaction occurs as an intramolecular reaction (cis mode) and the subsequent His–Asp–His phosphorelay reactions occur as intermolecular reactions (trans modes), in other words, the three-step phosphorelay of
ArcB proceeds in a cis-trans-trans mode.
Sinorhizobium meliloti FixL/FixJ, in vitro kinase assay
E.coli EvgS/EvgA,in vitro kinase assay -1
E.coli EvgS/EvgA,in vitro kinase assay -2
E.coli BarA/UvrY, in vitro kinase assay
E.coli ,PhoR/PhoB, in vitro kinase assay
E.coli EnvZ, in vitro kinase assay