TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/12902/
KW  - X-ray crystallography 
KW  -  ethylene 
KW  -  signal transduction 
KW  -  Arabidopsis thaliana 
KW  -  kinase
TI  - Structural studies of the Arabidopsis thaliana ethylene signal transduction pathway
Y1  - 2011///
ID  - heidok12902
A1  - Mayerhofer, Hubert
AV  - public
N2  - Since ethylene was first recognized as a phytohormone, the scope of its profound and multi-faceted impact on plant growth and development has been continuously growing. In Arabidopsis thaliana the response to ethylene is regulated by a group of five receptors (ETR1, ETR2, ERS1, ERS2 and EIN4), which are located in the endoplasmic reticulum membrane. Ethylene is bound by the membrane-embedded N-terminal part, which is followed by a GAF domain. The remaining C-terminal, cytosolic domains resemble the classical bacterial two-component system consisting of a histidine kinase (HK) and in some receptors a receiver domain. Constitutive triple response 1 (CTR1), whose kinase domain bears most resemblance to the RAF family of Ser/Thr protein kinases, directly interacts with the ethylene receptors and thus links signal reception to the intracellular signalling pathway. Therefore, this signalling pathway presents the interesting case, wherein a two-component signalling system manipulates a MAPKKK and possibly a MAPKKK signalling cascade. Still, the question of receptor deactivation by ethylene and the ensuing signal transduction to CTR1 remain unanswered. A number of constructs comprising domains of the receptors as well as the kinase domain of CTR1 were expressed and purified. The dimerization domain (DHp) of ERS1 could be crystallized and solved by MAD to 1.9 Å resolution. The domain is structurally similar to other domains of this family, normally found in bacteria, consisting of a homodimer forming a coiled-coil and a four-helix bundle. Different to the previously available structures a larger portion of the N-terminal coiled-coil could be determined. When the DHp domain structure is compared with other HKs it is most similar to the phosphatase-competent state, one of the three activities attributed to HKs. A trans-phosphorylation mechanism in the dimer is predicted by topological arrangement of the structure. In addition SAXS data of the cytoplasmic part of ETR1 was collected and a model of the domain architecture is presented, showing their relative location with respect to each other. A different location of the receiver domain compared to a recent bacterial structure is suggested. In addition the three-dimensional structures of the active, tri-phosphorylated and the unphosphorylated, inactive kinase domain of CTR1 in complex with staurosporine were determined at 3.0 Å and 2.5 Å resolution, respectively. They illustrate the conformational rearrangements that form the basis of activity regulation. The active kinase domain forms back-to-back dimers in solution, while the unphosphorylated kinase is a monomer. The back-to-back dimer interface is virtually identical to the one found in B-RAF and a number of mutants were identified interfering with the dimerization and also affecting the kinase activity. Furthermore the effects of activation loop phosphorylation on the activity were explored. The results strongly suggest another layer of activity regulation of CTR1 through dimerization in vivo. Steric restraints further indicate regulation of kinase activity across dimers with a 'front-to-front' activation interface, which points to CTR1 mediated ethylene receptor crosstalk generating a continuous head-to-tail oligomer of kinase domains.  
ER  -