Preparation, crystallization and preliminary
structural analysis of AHP2 protein, the signal transmitter from Arabidopsis thaliana
Oksana Degtjarik1,2,3, Radka Dopitova1, Sandra Puehringer5,
Manfred S. Weiss5, Lubomir Janda1, Jan Hejatko1 and Ivana Kuta-Smatanova2,4
1 Masaryk
University, Central European
2
University 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
3
University 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
Histidine-containing phosphotranfer proteins from Arabidopsis thaliana (AHP1-5) function as a signal
transmitters between sensor histidine kinases and response regulators via multistep
phosphorelay (MSP). AHP proteins mediate and
potentially integrate various MSP signalling pathways (e.g. cytokinin,
ethylene, osmosensing). However, structural
information about AHP proteins and its importance in the MSP signalling is
scarce.
To get insights into structural determinants of AHP-mediated signalling,
the coding sequence of AHP2 protein was cloned and purified to the homogeneity.
To enhance its crystallization behaviour, AHP2 was transferred to the buffer optimal
for its thermodynamic stability prior to crystallization. The obtained crystals
diffracted up to 2.5 Å (BESSY, Berlin) and show significant anisotropic behaviour. AHP2
structure was determined by SIRAS protocol using the anomalous signal of
the Lutetium.
According to the preliminary results AHP2 consists of a
four-helix bundle with the phophorylated His residue
in the center of the second helix, thus representing
the conserved core common for all known HPt
structures. The final structure refinement is currently being in progress.
This work was supported by grants: CZ.1.05/1.1.00/02.0068,
CZ.1.05/2.1.00/01.0024), P305/11/0756, AV0Z60870520.