EFFETCS OF MAGNESIUM BINDING AND BERYLLOFLUORIDATION ON A COMPONENT OF THE CYTOKININ SIGNALING PATHWAY STUDIED BY NUCLEAR MAGNETIC RESONANCE

Olga Třísková¹, Blanka Pekárová², Veronika Motáčková¹, Lukáš Žídek¹, Vladimír Sklenář¹, Jaromír Marek² ,Lubomír Janda²

¹National Centre for Biomolecular Research and ²Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 61137 Brno, Czech Republic

Cytokinins are important regulators of intrinsic developmental programs leading to de- and re-differentiation of plant cells. The cytokinin signal transduction seems to be mediated via multistep phospohorylation, similar to the action of bacterial two-component signalling systems. A soluble receiver domain of CKI1 receptor histidine kinase of Arabidopsis thaliana has been investigated by nuclear magnetic resonance (NMR) in this study. The domain was expressed in Escherichia coli and labeled with stable isotopes (C-13, N-15). Resonance frequencies have been assigned using standard strategy and conformational changes were monitored by running 2D ¹H-¹⁵N HSQC spectra. Effect of Mg(2+) has been studied in a series of titration experiments and the most significantly affected residues were identified using secondary chemical shift mapping. Activation of the protein, which cannot be studied in real time due to a short life-time of the activated form with phosphorylated aspartate, was investigated in an artificial system with phosphate replaced by beryllofluoride. In both cases, the observed chemical shift changes were mapped on a recently solved X-ray structure of the non-phosphorylated protein. In addition to the structural studies, molecular motions were investigated by N-15 NMR relaxation experiments. Series of relaxation spectra were obtained for free, Mg(2+)-bound, and beryllofluorinated protein and interpreted in terms of the Lipari-Szabo model-free approach. The observed changes are discussed in context of the X-ray structure of the free protein.

Acknowledgment:

This work was supported by the Grants MSM0021622413, MSM0021622415, LC06034, and LC06030 of the Ministry of Education, Youth, and Physical Culture of the Czech Republic.