Interaction of CheY2 and CheY2‐P with the cognate CheA kinase in the chemosensory‐signalling chain of Sinorhizobium meliloti

Abstract

SummaryAn unusual regulatory mechanism involving two response regulators, CheY1 and CheY2, but no CheZ phosphatase, operates in the chemotactic signalling chain of Sinorhizobium meliloti. Active CheY2-P, phosphorylated by the cognate histidine kinase, CheA, is responsible for flagellar motor control. In the absence of any CheZ phosphatase activity, the level of CheY2-P is quickly reset by a phospho-transfer from CheY2-P first back to CheA, and then to CheY1, which acts as a phosphate sink. In studying the mechanism of this phosphate shuttle, we have used GFP fusions to show that CheY2, but not CheY1, associates with CheA at a cell pole. Cross-linking experiments with the purified proteins revealed that both CheY2 and CheY2-P bind to an isolated P2 ligand-binding domain of CheA, but CheY1 does not. The dissociation constants of CheACheY2 and CheA-CheY2-P indicated that both ligands bind with similar affinity to CheA. Based on the NMR structures of CheY2 and CheY2-P, their interactions with the purified P2 domain were analysed. The interacting surface of CheY2 comprises its C-terminal b4-a4-b5-a5 structural elements, whereas the interacting surface of CheY2-P is shifted towards the loop connecting b5 and a5. We propose that the distinct CheY2 and CheY2-P surfaces interact with two overlapping sites in the P2 domain that selectively bind either CheY2 or CheY2-P, depending on whether CheA is active or inactive.

Sinorhizobium meliloti CheA Complexed with CheS Exhibits Enhanced Binding to CheY1, Resulting in Accelerated CheY1 Dephosphorylation

“…Proteins were cross-linked with glutaraldehyde (Serva, Germany) at a final concentration of 1% (wt/vol) as previously described (41). Briefly, reactions were carried out at protein concentrations of 0.7 or 1.0 M in 20 mM NaCl, 1 mM EDTA, 20 mM NaPO 4 , pH 7.5, or in 150 mM NaCl, 50 mM NaPO 4 , pH 8.0, at room temperature.…”

“…Surface plasmon resonance experiments were performed on a Biacore X instrument (GE Healthcare) essentially as described previously (41). A standard CM5 chip was loaded with purified His 6 -CheA/CheS (in 10 mM Na-acetate buffer, pH 4.2) according to the supplier's standard amino coupling protocol.…”

“…We used a chemical cross-linking assay to identify putative interaction partners of CheS. Recombinant CheY1, CheY2, and CheA were individually overexpressed in E. coli ER2566 and purified using the IMPACT system (41). Equimolar amounts of proteins (final concentration, 0.7 M) were cross-linked with 1% (wt/vol) glutaraldehyde, separated electrophoretically, and detected by immunoblotting using anti-CheS antibody.…”

“…S2 in the supplemental material). It is known from previous studies that individual structural elements of CheA can be stably expressed (12,17,41). Domains were defined by applying the published domain structure of E. coli CheA to S. meliloti CheA (23,56,57).…”

“…It has been proposed that S. meliloti CheY1 competes for phosphoryl groups from CheA-P with an apparent higher affinity than CheY2 (52). However, in vitro and in vivo binding analyses suggested that CheY1-CheA binding is much weaker than CheY2-CheA binding (41). In a previous study we established the apparent dissociation constant of CheY2 as 0.32 M, while the value determined for CheY1 was 170 M (41).…”