Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana.

Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD)) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg2+, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF3- on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg2+. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD), with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD), as was determined by both X-ray crystallography and NMRspectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.