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 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (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 CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the β3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the β3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the β3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the β3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic β3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.

• Klíčová slova
• X-ray crystallography, histidine kinase, nuclear magnetic resonance (NMR), protein dynamic, protein phosphorylation, receiver domain, relaxation dispersion,
• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• krystalografie rentgenová MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteinkinasy chemie   genetika   MeSH
• proteinové domény MeSH
• proteiny huseníčku chemie   genetika   MeSH
• receptory buněčného povrchu chemie   genetika   MeSH
• sekundární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CKI1 protein, Arabidopsis MeSH Prohlížeč
• ETR1 protein, Arabidopsis MeSH Prohlížeč
• proteinkinasy MeSH
• proteiny huseníčku MeSH
• receptory buněčného povrchu MeSH