Crystal structure of the receiver domain of the histidine kinase CKI1 from Arabidopsis thaliana

T. Klumpler, B. Pekárová, J. Marek, P. Borkovcová, L. Janda, J. Hejátko

Laboratory of Molecular Plant Physiology, Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Faculty of Science, Masaryk University

klumpler@sci.muni.cz

The crystal structure of the receiver domain of the histidine kinase CKI1 from Arabidopsis thaliana has been determined at a resolution of 2.0 Å.

Sensor histidine kinases (HKs) are members of the two-component (TC) signalling systems that mediate signal transduction in a broad spectrum of adaptive responses in bacteria [1]. A modified version of bacterial two-komponent (TC) signalling has been adapted by yeast and plants. In TC signalling in plants, the membrane-associated sensor HK interacts with a signalling molecule, which activates an intracellular HK domain and leads to autophosphorylation of its conserved histidine moiety. The downstream phosphorelay is initiated by a receiver domain (RD) of the HK. The RD transfers phosphate from a His to its own Asp and further transmits the signal via transphosphorylation to the His of a histidine-containing phosphotransfer (HPt) domain. The HPt proteins translocate the signal to the nucleus, where the phosphorylated histidine serves as a donor for the phosphorylation of a final phosphate acceptor, the Asp residue of the response regulator [2].

In the A. thaliana genome, genes encoding 11 HKs, 6 HPt proteins and 23 response regulators have been identified. A. thaliana HKs mediate discrete responses to various phytohormones (ethylene, cytokinin and abscisic acid) and osmosensing [3]. This suggests that the structure of the RD might contribute to the recognition of its interaction partners.

The sensor histidine kinase CKI1 was identified as an activator of a cytokinin-like response when overexpressed in hypocotyl explants of A. thaliana [4]. However, in contrast to the genuine cytokinin receptors of A. thaliana, AHK2, AHK3 and AHK4, CKI1 was found to be constitutively active in bacteria and yeast or A. thaliana protoplasts [5-6]. Thus, the specificity and the role of CKI1 in the TC signalling in A. thaliana remain unclear.

Crystals of the recombinant RD of the Arabidopsis HK CYTOKININ-INDEPENDENT1 (CKI1_RD) have been obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant and glycerol as a cryoprotectant. The crystals diffracted at beamline BW7B of the DORIS-III storage ring to approx. 2.4 Å. The diffraction has been improved significantly - to at least 2.0 Å - after applying of a non-water cryoprotectant. The crystals belong to space group C222₁ with unit-cell parameters a=54.46, b=99.82, c=79.94 Å, the asymmetric unit contains one molecule of the protein. The structure of CKI1_RD had been solved by a molecular-replacement method using an automated scheme for molecular replacement as implemented in MrBUMP v.0.4.1 in conjunction REFMAC as the refinement program. An unambiguous solution was found using the bacterial response-regulator protein CheY [7] as a search model. Initial R value of 0.54, which decreased to R = 0.413 and Rfree = 0.426 after 30 cycles of REFMAC refinement. The quality of the map generated with this result was good enough to allow successful application of the autobuild regime of ARP/wARP.

The three-dimensional structure of A. thaliana CKI1_RD shows the conformational conservation of receiver proteins, such as CheY, CheB, ETR_RD. CKI1_RD is a single domain protein folded in a (β/α)₅ manner with a central β-sheet formed from five β-strands and surrounded by sides by two and three α-helices. The catalytic aspartate residue is located on the carboxyl terminus of the central β3-strand, in a cavity formed by loops L1, L5 and L7 loops. All major conformational differences between receiver proteins are located in the loops, which supposedly form a docking interface for the ineracting partners.

References

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