Preparation and crystallization of AHP2 protein, the signal transmitter from Arabidopsis thaliana

 

Radka Dopitová1,*, Oksana Degtjarik2,3*, Tatsiana Holubeva2,3, Ivana Kutá-Smatanová2,4, Jan Hejátko1 and  Lubomír Janda1

 

 

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, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses and School 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, Inst. of Nanobiology and Structural Biology GCRC, Zámek 136, CZ-373 33 Nové Hrady

*these authors contributed equally

 

Histidine phosphotranfer proteins (HPts) from Arabidopsis thaliana (AHP1-5) mediate signal transduction downstream from sensor histidine kinases (HK) to subsequent phospho-accepting response regulators (RR) via so called multistep phosphorelay (MSP). AHP proteins are involved in and potentially integrate various MSP signalling pathways (e.g. cytokinin, ethylene, osmosensing). However, our knowledge on structure of AHP proteins and its importance in the MSP signalling is scarce.

Here we report on expression, purification and preliminary crystallization of AHP2. Expression of AHP2 have been optimized and the sample quality was improved by using reducing conditions during purification procedure. Crystallization conditions were found, but crystals diffracted only to 6 Å resolution (X-ray source at Institute of Organic Chemistry and Biochemistry AS ČR, v.v.i. and X13 beamline at Dessy, Hamburg). Further screening of optimal buffer conditions using differential scanning fluorimetry and limited proteolysis approach resulted in crystals diffracting to 3 Å resolution (Bessy, Berlin). High-quality crystals for data collection are currently being produced.

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.