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
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.
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
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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.