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.
Korzhnev D. M., Billeter M., Arseniev A. S., and Orekhov V. Y. (2001) NMR studies of Brownian tumbling and internal motions in proteins. Prog. Nucl. Magn. Reson. Spectrosc. 38, 197–266
Lipari G., and Szabo A. (1982) Model-free approach to the interpretation of nuclear magnetic-resonance relaxation in macromolecules. 1. Theory and range of validity. J. Am. Chem. Soc. 104, 4546–4559
Lipari G., and Szabo A. (1982) Model-free approach to the interpretation of nuclear magnetic-resonance relaxation in macromolecules. 2. Analysis of experimental results. J. Am. Chem. Soc. 104, 4559–4570
Halle B., Andersson T., Forsén S., and Lindman B. (1981) Protein hydration from water oxygen-17 magnetic-relaxation. J. Am. Chem. Soc. 103, 500–508
Loria J. P., Rance M., and Palmer A. G. (1999) A relaxation-compensated Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy. J. Am. Chem. Soc. 121, 1331–1332
Krug M., Weiss M. S., Heinemann U., and Mueller U. (2012) XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS. J. Appl. Crystallogr. 45, 568–572
Bodenhausen G., and Ruben D. J. (1980) Natural abundance N-15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Lett. 69, 185–189
Sklenář V., Piotto M., Leppik R., and Saudek V. (1993) Gradient-tailored water suppression for 1H-15N HSQC experiments optimized to retain full sensitivity. J. Magn. Reson. Ser. A 102, 241–245
Grzesiek S., and Bax A. (1992) Correlating backbone amide and side-chain resonances in larger proteins by multiple relayed triple resonance NMR. J. Am. Chem. Soc. 114, 6291–6293
Wittekind M., and Mueller L. (1993) HNCACB, a high-sensitivity 3D NMR experiment to correlate amide-proton and nitrogen resonances with the α-carbon and β-carbon resonances in proteins. J. Magn. Reson. Ser. B 101, 201–205
Muhandiram D. R., and Kay L. E. (1994) Gradient-enhanced triple-resonance 3-dimensional NMR experiments with improved sensitivity. J. Magn. Reson. Ser. B 103, 203–216
Clore G. M., Szabo A., Bax A., Kay L. E., Driscoll P. C., and Gronenborn A. M. (1990) Deviations from the simple 2-parameter model-free approach to the interpretation of N-15 nuclear magnetic-relaxation of proteins. J. Am. Chem. Soc. 112, 4989–4991
Luz Z., and Meiboom S. (1963) Nuclear magnetic resonance study of protolysis of trimethylammonium ion in aqueous solution–order of reaction with respect to solvent. J. Chem. Phys. 39, 366