Conformational dynamics are a key factor in signaling mediated by the receiver domain of a sensor histidine kinase from Arabidopsis thaliana
2017
Multistep phosphorelay (MSP) cascades mediate responses to a
wide spectrum of stimuli, including plant hormonal signaling,
but several aspects of MSP await elucidation. Here, we provide
first insight into the key step of MSP- mediated
phosphotransfer in a eukaryotic system, the phosphorylation of
the receiver domain of the histidine kinase
CYTOKININ-INDEPENDENT 1 (CKI1(RD)) from Arabidopsis thaliana.
We observed that the crystal structures of free, Mg2+-bound,
and beryllofluoridated CKI1(RD) (a stable analogue of the
labile phosphorylated form) were identical and similar to the
active state of receiver domains of bacterial response
regulators. However, the three CKI1(RD) variants exhibited
different conformational dynamics in solution. NMR studies
revealed that Mg2+ binding and beryllofluoridation alter the
conformational equilibrium of the beta 3-beta 3 loop close to
the phosphorylation site. Mutations that perturbed the
conformational behavior of the beta 3-beta 3 loop while keeping
the active- site aspartate intact resulted in suppression of
CKI1 function. Mechanistically, homology modeling indicated
that the beta 3 beta 3 loop directly interacts with the ATP-
binding site of the CKI1 histidine kinase domain. The
functional relevance of the conformational dynamics observed in
the beta 3-beta 3 loop of CKI1(RD) was supported by a
comparison with another A. thaliana histidine kinase, ETR1. In
contrast to the highly dynamic beta 3- beta 3 loop of CKI1(RD),
the corresponding loop of the ETR1 receiver domain (ETR1(RD))
exhibited little conformational exchange and adopted a
different orientation in crystals. Biochem-ical data indicated
that ETR1(RD) is involved in phosphorylation-independent
signaling, implying a direct link between conformational
behavior and the ability of eukaryotic receiver domains to
participate in MSP.
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