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 Institute of Technology (CEITEC), Žerotínovo nám. 9, CZ-60177 Brno, Czech Republic

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.