Structural characterization of the members of the multistep signaling system from A. thaliana

 

Oksana Degtjarik1,2,3, Radka Dopitova1, Sandra Puehringer5, Blanka Pekarova1,
Manfred S. Weiss5, Lubomir Janda1,  Jan Hejatko1 and Ivana Kuta-Smatanova2,4

 

1Masaryk University, Central European Institute of Technology (CEITEC), Žerotínovo nám. 9, CZ-60177 Brno, Czech Republic

2University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, CENAKVA and Institute of Complex Systems, Zámek 136, CZ-373 33 Nové Hrady

3University of South Bohemia in České Budějovice, Faculty of Science, Branišovská 31, CZ-37005 České Budějovice

4Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Zámek 136, CZ-373 33 Nové Hrady

5Helmholtz-Zentrum Berlin für Materialien und Energie BESSY-II, Albert-Einstein-Straße 15, 12489 Berlin, Germany

degtjarik@frov.jcu.cz

 

Introduction

Multistep signaling system (or multistep phosphorelay) plays very important role in sensing/response mechanisms in higher plants. It consists of three components: a sensory histidine kinase, which catalyses the autophosphorilation of a His residue and subsequently transfers the phosphoryl group to Asp residue of its internal receiver domain. Then, the signal is passed downstream to a His residue of histidine phosphotransfer protein (Hpt) and finally delivered to an Asp residue of receiver domain of the corresponding response regulator, which subsequently interacts with target proteins or acts as a transcription factors [1,2]. In Arabidopsis, MSP signaling regulates various key processes, such as osmosensing, hormone signaling and gametogenesis [3]. Arabidopsis genome encodes 8 histidine kinases and 5 canonic Hpt (AHP1-AHP5) [4]. The goal of this work is to get insights into structural determinants of AHP-mediated signaling by the example of receiver domain of histidine kinase 1 (CKI1, PDB code 3MMN) and AHP2.

Experimental section

To achieve our goal AHP2 gene was cloned and transformed into E. coli expression strain. The protein was expressed and purified in 2 steps: by metal chelate affinity chromatography followed by anion-exchange chromatography. Initial screening of crystallization conditions was performed by sitting drop vapor diffusion method using different commercial screens. Optimization of the discovered conditions included the screening of pH, incubation temperature, type and concentration of the precipitant. The optimal buffer composition for crystallization was determined using a thermal shift assay. Diffraction data were collected on BL14.2 at the BESSY II storage ring and processed with XDSAPP. Structure of AHP2 was solved with SIRAS protocol, using anomalous signal of the Lutetium. Substructure solution, phasing, density modification and initial model building was carried out using the programs SHELXC/D/E via the graphical user interface HKL2MAP. Side chains were assigned using the autobuild/refine protocol in BUCCANEER. An anomalous difference Fourier electron density map was calculated using CCP4 program. Structure refinement was carried out using REFMAC5 and iterated with manual model building in COOT.

            Protein-protein rigid body docking with AHP2 and CKI1rd was performed by GRAMM-X web server followed by molecular dynamic simulations using GROMACS software. The simulation was performed at 300K for 100ns.

 

Results and discussion 

The HPt AHP2 was cloned, overexpressed and purified to >95 % purity. The initial crystallization conditions for AHP2 were identified in condition No. 20 of Crystal Screen 2 (Hampton Research) consisting of 0.1 M MES buffer pH 6.5 and 1.6 M MgSO4. Small tetragonal crystals were grown in 2 days at 298 K, but they did not diffract X-rays beyond 6Å resolution. To enhance the crystallization behaviour, AHP2 was transferred to the buffer optimal for its thermodynamic stability prior to crystallization based on the results of the thermal shift assay. As the most stabilizing buffer for AHP2 50 mM imidazole, pH 8.0 supplemented with 5 mM DTT was identified. The protein was then transferred to this buffer prior to crystallization and simultaneously, the crystallization temperature was lowered to 4°C. This optimization resulted in formation of crystals with maximum dimensions of approximately 0.35 x 0.2 x 0.1 mm. which diffracted up to 2.5 Å and show significant anisotropic behaviour.

The structure of AHP2 was solved using SIRAS protocol using anomalous signal of the Lutetium. AHP2 protein represents α-helical bundle, comprising of four short and two long helices. Short helices form a central core of the protein with the conserved His residue carrying the phosporyl group situated in the middle of the third α-helix.

In order to gain insight into the structural features, underlying AHP2-CKI1rd interaction, molecular-dynamics simulations were carried out. Simulations were performed for 100 ns and show the stability of protein-protein complex. The key residues, responsible for the AHP2-CKI1rd interaction, were identified and reveal strong protein-protein binding. The analysis of the obtained data is currently in progress.

References         

1. Dortay, H., Mehnert, N., Burkle, L., Schmulling, T. & Heyl, A. (2006). FEBS. 273, 4631-4644.

2. Schaller, D., Alonso G. E., Ecker J. M. & Kieber J. R., J. J. (2006). Plant Cell. 18, 3073-3087.

3. Stock, A. M., Robinson, V. L. & Goudreau, P. N. (2000). Annu. Rev. Biochem. 69, 183-215.

4. Suzuki, T., Sakurai, K., Imamura, A., Nakamura, A., Ueguchi, C. & Mizuno, T. (2000). Biosci Biotechnol Biochem. 64, 2486-2489

Acknowledgements

The work was supported by CEITEC – Central European Institute of Technology CZ.1.05/1.1.00/02.0068, GAČR 521/09/1699, P305/11/0756, ME ČR ME09016, CZ.1.05/2.1.00/01.0024, by the AS ČR AV0Z60870520.