The following devices are available in the group.
Molecular recognition
Extracellular signal transduction
In addition to its important cellular function in metabolism, ATP also serves as an extracellular signalling substance. It acts on P2X receptors, which are ligand-gated ion channels, and P2Y receptors, which are G protein-coupled receptors (GPCR). These signalling pathways via ATP and other nucleotides are called purinergic signalling pathways.
Extracellular nucleotides influence a variety of short-term physiological processes, including exocrine and endocrine secretion, immune responses, inflammation, nociceptive mechanosensory transduction, platelet aggregation and endothelium-mediated vasodilation. Long-term (trophic) processes are cell proliferation, differentiation, migration and death. These play a major role in developmental processes and also in carcinogenesis.
As with any signalling molecule, its effect must be terminated over time. A number of extracellular nucleotidases (ecto-nucleotidases) are involved in the hydrolysis of nucleotides. The NTPDases dephosphorylate ATP via ADP to AMP. The ecto-5'-nucleotidase (e5NT, eN, CD73) catalyses the hydrolysis of AMP to adenosine. In addition to the receptors, the ecto-nucleotidases are also attractive targets for drug development, for example in cancer immunotherapy. Extracellular ATP has an immunostimulatory effect. Adenosine, on the other hand, acts as an agonist on G-protein-coupled P1 receptors and has an immunosuppressive effect.
The NTPDases hydrolyse ATP to ADP and ADP to AMP, in each case by cleaving off the terminal phosphate group. In humans, there are eight homologous NTPDases, of which NTPDases 1-3 and 8 are extracellularly anchored to the cell membrane. NTPDases 4-7 are present intracellularly in cell organelles such as the Golgi and the ER and are partly involved in the hydrolysis of UDP, which is formed from UDP-glucose during the glycosylation of proteins.
We have elucidated the structure of NTPDase2 as the first spatial structure of this enzyme family (Figure left). The enzyme consists of two protein domains and the ATP binds in the cleft between these domains. A structural analysis with AMPPNP as a non-hydrolysable substrate analogue to ATP showed the binding of the substrate and led to a model for the reaction mechanism in which a water nucleophile attacks the terminal phosphate group (Figure right). We also elucidated the structure of NTPDase1 (CD39), which is mainly involved in the degradation of immunostimulatory ATP.
Later, we studied structures of NTPDases from unicellular pathogenic microorganisms such as Toxoplasma gondii and Legionella pneumophila. These pathogens are supposed to express the NTPDases to suppress the host's immune response.
Currently, our research focuses on intracellular NTPDases and the development of NTPDase inhibitors.
Ecto-5'-nucleotidase, also known as CD73, is part of the purinergic signalling cascade by hydrolysing AMP to adenosine. This turns on the adenosine signalling pathway via P1 receptors. E5NT is involved in the development of chronic pain, hypoxia and inflammation. In addition, CD73 has been shown to be overexpressed in many cancers for tumour promotion and metastasis. CD73 inhibitors are of interest for cancer immunotherapy (see drug development information on this website).
Through studies on the human enzyme and the homologous 5'-nucleotidase from E. coli, we have intensively investigated the unique domain movement of the enzyme and the catalytic mechanism. CD73 is a dimeric enzyme. The domain movement allows binding of the substrate to the open form and release of the products (figure left). Only in the closed state the active site, which lies between the two protein domains of the enzyme, is complete and catalytically active. The homologous monomeric 5'-nucleotidase from E. coli hydrolyses not only AMP but also ATP and ADP (and other nucleotides). A crystallographic determination of the binding mode of the non-hydrolysable ADP analogue AMPCP revealed a catalytically competent binding mode in which the terminal phosphate group is coordinated to one of the two zinc ions, while the other metal ion activates a water nucleophile (top right figure). We postulate that a similar binding mode of the phosphate group of AMP leads to hydrolysis of AMP.
CD73, on the other hand, is inhibited by ATP and ADP. The observed binding mode of AMPCP to CD73 is indeed an inhibitory binding mode (bottom right figure). The terminal phosphate group bridges the two metal ions and the water nucleophile has been displaced. AMPCP derivatives have been shown to be excellent inhibitors of CD73. The investigation of the binding mode of these derivatives is part of the current research of our group and is described in more detail in the section on structure-based inhibitor development.
Adhesion G protein-coupled receptors (aGPCR) are a large receptor family that has only been partially investigated with regard to their biochemical, structural and functional properties. The aim of the project is to characterise the spatial structures of the receptors and their interactions with binding partners by X-ray structural analysis. Such investigations are the basis for understanding the molecular function of the receptors and for structure-based development of potential drugs that act on these receptors.
For this purpose, the receptors and single domains are expressed primarily in HEK cells. Milligram quantities of highly pure protein must be produced for structural and biophysical investigations. The following processes in the mode of action of the receptors are primarily studied: The binding of ligands to the extracellular binding domains and the conformational changes induced by this, the function of the autoproteolytic GAIN domain and the interaction between the GAIN domain and the transmembrane part, which is probably conserved in all aGPCR.
Adhesion GPCRs are a current research focus in Leipzig. This research is funded by a DFG research group and by the Collaborative Research Centre CRC1423.
Adhesion G protein-coupled receptors (aGPCR) are a large receptor family that has only been partially investigated with regard to their biochemical, structural and functional properties. The aim of the project is to characterise the spatial structures of the receptors and their interactions with binding partners by X-ray structural analysis. Such investigations are the basis for understanding the molecular function of the receptors and for structure-based development of potential drugs that act on these receptors.
For this purpose, the receptors and single domains are expressed primarily in HEK cells. Milligram quantities of highly pure protein must be produced for structural and biophysical investigations. The following processes in the mode of action of the receptors are primarily studied: The binding of ligands to the extracellular binding domains and the conformational changes induced by this, the function of the autoproteolytic GAIN domain and the interaction between the GAIN domain and the transmembrane part, which is probably conserved in all aGPCR.
Adhesion GPCRs are a current research focus in Leipzig. This research is funded by a DFG research group and by the Collaborative Research Centre CRC1423.