Structural characterization of Arabidopsis thaliana ethylene signaling molecules and the non-ribosomal peptide synthetase from Planktothrix agardhii
Plants employ a complex network of signaling pathways to regulate developmental processes and to mediate the responses to both environmental and biological stress factors. Ethylene is one of the key plant hormones involved in controlling this network, which has made it and its signaling pathway a target of intense research for several decades. In the model plant Arabidopsis thaliana, the plant hormone is detected by a group of five receptors (ETR1, ERS1, ETR2, ERS2 and EIN4) that resemble the sensor histidine kinases of bacterial two-component system. The main aim in this thesis study was the expression and purification of the full-length ETR1 for structural studies to gain insights into the initial steps in ethylene signaling. The FL ETR1 was successfully expressed in baculovirus expression vector system but the isolation of the receptor from the membrane was hampered. In addition to the FL ETR1, the cytosolic portion of the receptor was studied using Small Angle X-ray Scattering. The resulting SAXS model had the expected dimeric arrangement. EDR1 from A. thaliana is a CTR1-like MAPKKK that is involved in regulating disease resistance responses, cell death and also ethylene-induced senescence. It possesses an N-terminal regulatory domain and C-terminal catalytic domain wit Ser/Thr kinase activity. As EDR1 has been shown to autophosphorylate in trans, the mechanism of this was studied using X-ray crystallography. A crystal structure for the catalytically inactive kinase domain of EDR1 (EDR1-D792N) was obtained in the presence of the ATP substrate analog AMP-PNP. The asymmetric unit contained two molecules, one of which surprisingly was in an active-like conformation. Furthermore, the active-like EDR1-D792N molecule was found to form an authentic trans-autophosphorylation complex with the inactive monomer from the adjacent asymmetric unit. In addition to the plant defense signaling proteins, an adenylation (A) domain from cyanobacterial non-ribosomal peptide synthetase (NRPS) was studied. NRPSs are large multidomain enzymes that are found from a number of fungal and bacterial species and catalyze the ribosome-independent assembly of biologically active peptides with diverse composition and function. The A domain plays a central role in the NRPS system as it recognizes and activates the amino acid, which is incorporated into the growing peptide. The A domain ApnA A1 from the Anabaenopeptin synthetase of Planktothrix agardhii is an interesting member of its class as it has an unusual ability to activate two very distinct amino acids (arginine and tyrosine). Structural studies on this enzyme were performed to elucidate its bi-specificity. Based on the solved ApnA A1 structures, two active site residues with a crucial role in the substrate binding were identified. The mutation of these residues led to enzyme variants, which were mono-specific for either tyrosine or arginine, or in some instances were able to activate L-tryptophan. Additionally a number of ApnA A1 mutants were shown to activate unnatural amino acids (4-fluorophenylalanine and 4-azidophenylalanine). A final peptide product with an unnatural amino acid incorporated, could possibly have useful industrial or pharmaceutical applications.
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