SUMMARYMultistep 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 Mg 2+ , the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF 3 ) on CKI1 RD in solution, and determined the crystal structure of free CKI1 RD and CKI1 RD in a complex with Mg 2+ . 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 NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.

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