Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana

Blanka Pekárová^1,3,*, Olga Třísková^2,3, Tomáš Klumpler^1,3,*, Jakub Horák^1,3, Radka Dopitová^1,3, Séverine Jansen^2,3, Lukáš Žídek^2,3, Jan Hejátko^1,3, Lubomír Janda^1,3

Department of Functional Genomics and Proteomics, Institue of Experimental Biology¹ and National Centre for Biomolecular Research², Masaryk University, Kotlářská 2, CZ-61137 Brno, Czech Republic;

Central European Institute of Technology, Masaryk University, Žerotínovo nám. 9, CZ-60177 Brno, Czech Republic³

Multistep phosphorelay (MSP) pathway mediate sensing of wide spectrum of signals in plants, including hormonal (cytokinin, ethylene, abscisic acid) and abiotic stress regulation. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-5) to nuclear response regulators. In contrast to bacteria, MSP protein interactions in plants are supposed to be rather non-specific.

A C-terminal receiver domain of histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1_RD) has been investigated in this study. Using both in vivo and in vitro assays we have found that CKI1_RD is responsible for recognition of CKI1 downstream signaling partners and specifically interacts with AHP2, 3 and 5 with different affinities. Effects of Mg²⁺, the cofactor necessary for signal transduction, and phosphorylation-mimicking BeF₃^- on CKI1_RD in solution were studied by NMR. Finally, crystal structures of free CKI1_RD and CKI1_RD in a complex with Mg²⁺ were determined. Magnesium binding induces rearrangement of some residues around the active site of CKI1_RD which affects its activity and specificity.

Supported by Grants Nos. 204/08/H054, 525/07/1069, 521/09/1699, P503/10/217, P305/11/0756, MSM0021622413, MSM0021622415, LC06034, EAST-NMR, and 205872, POSTBIOMIN - FP7-REGPOT-2007-1.