Müller-Dieckmann Team
X-ray crystallography, high-throughput crystallisation
Ribbon representation of Rv0066c (Icd2) of M. tuberculosis. Shown is a dimer of Icd2 with a two-fold axis in the image plane. α-helices are in red and β-strands are in blue. Each monomer consists of 745 amino acids. Substrate and NADPH are bound in the large cleft between domains.
Previous and current research
Structural biology: Our goal is to understand the molecular principles of specific disease related proteins and those of signal transduction across membranes. For the latter, we have chosen a relatively simple yet well-characterised system from the model plant Arabidopsis thaliana. Here, we try to structurally characterise a group of functionally related, membrane bound receptors and their interaction with a common cytosolic downstream target, a protein kinase. The system regulates the response to the simple phytohormone ethylene.
Ethylene is gaseous hormone in plants which regulates a multitude of processes, ranging from seed germination and fruit ripening to leaf abscission and organ senescence. Signal transduction involving ethylene is initiated by five receptors in A. thaliana. The functional unit of the receptors are disulfide linked dimers. All receptors share a small, highly conserved ER membrane embedded domain, which contains a single ethylene binding site. The cytosolic domain structure is related to bacterial two-component systems (TCS). In contrast to classical TCS, the immediate downstream target of the receptors, however, is CTR1, a Raf-like Ser/ Th r protein kinase. Thus, this signalling pathway presents an interesting case, wherein a two component signalling system manipulates a MAP kinase kinase kinase (MAP3K) and possibly a MAP3K signalling cascade.
The mechanism of signal transduction from eukaryotic two-component systems to classical MAPK signalling pathways remains unclear. We have produced a variety of constructs of the different signal receptors as well as of the cognate protein kinase. These domains and their complexes are analysed by X-ray crystallography and X-ray small angle scattering with the goal to functionally characterise the early steps of ethylene signalling through inter- and cross-domain activation of its components.
High-throughput crystallisation: EMBL Hamburg operates one of Europe’s largest high-throughput crystallisation facilities, which is open to the general user community. Currently, users come from over 20 different nations. Since 2009 our platform is part of the European FP7 initiative P-Cube, which supports access to advanced infrastructures. More information on this initiative is available at: www.p-cube.eu.
Future projects and goals
We have functionally and structurally characterised the active and inactive forms of the protein kinase domain of CTR1, as well as two sub-domains of the cytosolic ethylene receptor. Our next goals are the characterisation of larger parts of the receptor, including the full-length construct. We are also working on complexes of CTR1 with the receptors domains. The latter requires an interdisciplinary approach due to transient complex formation and unpredictable crystallisability. To this end, we will use a combination protein characterisation tools, X-ray crystallography and SAXS.