Expression, purification and crystallization of the intracellular domains of the ethylene receptor ETR1 from Arabidopsis thaliana
| Autoři | |
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| Rok publikování | 2015 |
| Druh | Další prezentace na konferencích |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | ETHYLENE RESPONSE 1 (ETR1) is a membrane-bound receptor from Arabidopsis thaliana involved in ethylene signalling. ETR1 possesses all of the sequence motifs of canonical histidine kinase (HK) domains and also reveals HK activity [1, 2]. HK activity and a presence of a C-terminal receiver domain (RD) may allow for cross-talk between ethylene signalling and multistep phosphorelay (MSP) in Arabidopsis. HK activity is regulated by the presence of divalent ions. Calcium and magnesium (Mg2+) ions are inhibiting HK activity of ETR1. On the other hand manganese (Mn2+) ion causes both HK and serine/threonine activity of ETR1. This mechanism of ETR1 could play a role as a signal switch between canonical CTR1/EIN2/EIN3 cascade of ethylene signalling and MSP pathway in A. thaliana. The main objective of our work is to elucidate structural aspects and HK activity of ETR1 in ethylene/MSP cross-talk. In order to achieve our goal we started with expression of intracellular domains of ETR1 in E. coli expression system and purification using affinity and gel filtration chromatography. We prepared pure, soluble proteins of dimerization, catalytic and receiver domains and we are currently optimising the kinase assay to prove functional activity of these domains. To investigate the structural changes induced by the binding of Mg2+ or Mn2+ ions to RD, X-ray crystallography was used. So far, crystals of RD (soaked or co-crystallized with Mg2+ or Mn2+ ions) were obtained using the procedure described previously [3] and the diffraction data were obtained at 2.5 A - 2.8 A resolution. In order to elucidate the effect of ions on RD, crystals diffracting to a better resolution are needed. Further optimization of crystallization conditions (including the temperature, crystallization technique or the presence of additives) is therefore currently ongoing. To overcome the size limits of NMR measurements, we are going to employ intein technology [4]. For that we prepared intein-containing DNA constructs of ETR1 domains. Now we are optimising expression and purification of intein-fused proteins. [1] Gamble et al. (1999), Proc. Natl. Acad. Sci. USA, 95:7825-7829. [2] Moussatche & Klee. (2004), J. Biol. Chem., Vol. 279, No. 47, 48734-48741. [3] Grantz et al. (1998), Acta Crystallogr D Biol Crystallogr. 54, 690-2. [4] Volkmann & Iwai, (2010), Mol. BioSyst., Vol. 6, 2110-2121. |
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