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.

In BriefThe oligomerization (and particularly dimerization) of Hsp70 proteins plays an important role in their chaperoning activities.Here, we report that human stress-inducible Hsp70 possesses the highest propensity among analyzed Hsp70 homologs to form dimers in the presence of ATP. ATP-bound Hsp70 assembles in solution as an antiparallel dimer closely resembling the dimeric structures captured in DnaK and BiP crystals. ATP-dependent Hsp70 dimerization is necessary for efficient Hsp40 interaction and is differentially affected by TPR cochaperone binding. Graphical Abstract Highlights• Hsp70 homologs differ in their oligomeric properties in the presence of ATP.• Human inducible Hsp70 forms ATP-dependent anti-parallel dimers with high propensity.• Dimerization of ATP-bound Hsp70 is required for effective Hsp70-Hsp40 interaction.• ATP-dependent interaction with Tomm34 TPR cochaperone disrupts Hsp70 dimer.