We explore the principles of molecular recognition in biological systems using methods of structure analyses. Molecular interactions not only determine the extraordinary specificity of enzymes or the activation of receptors in biological signal transduction. They are also the key to structure-based drug development.
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.
Publication
- H. Zimmermann, M. Zebisch, N. Sträter (2012)
Cellular function and molecular structure of ecto-nucleotidases.
Purinergic Signalling 8, 437-502. doi
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.
Publications
NTPDases from mammals
- Zebisch M, Sträter N.
Characterization of Rat NTPDase1, -2, and -3 ectodomains refolded from bacterial inclusion bodies.
Biochemistry 2007; 46, 11945-56. doi - Zebisch M, Sträter N.
Structural insight into signal conversion and inactivation by NTPDase2 in purinergic signaling.Proc Natl Acad Sci U S A. 2008; 105(19): 6882-7. doi
-
Zebisch M, Krauss M, Schäfer P, Sträter N.
Crystallographic evidence for a domain motion in rat nucleoside triphosphate diphosphohydrolase (NTPDase) 1.
J Mol Biol. 2012; 415(2):288-306. doi
Microbial NTPDases
- Krug U, Zebisch M, Krauss M, Sträter N.
Structural insight into activation of Toxoplasma gondii nucleoside triphosphate diphosphohydrolase by disulfide reduction.
J. Biol. Chem. 2012; 287, 3051-3066. doi - Krug U, Totzauer R, Sträter N.
The crystal structure of Toxoplasma gondii nucleoside triphosphate diphosphohydrolase 1 represents a conformational intermediate in the reductive activation mechanism of the tetrameric enzyme.
Proteins 2013; 81, 1271-1276. doi - Zebisch M, Krauss M, Schäfer P, Lauble P, Sträter N.
Crystallographic snapshots along the reaction pathway of nucleoside triphosphate diphosphohydrolases (NTPDases).
Structure 2013; 21, 1460-1475. doi - Krug U, Totzauer R, Zebisch M, Sträter N.
The ATP/ADP substrate specificity switch between Toxoplasma gondii NTPDases1 and -3 is caused by an altered substrate base binding mode.
ChemBioChem 2013, 14, 2292-2300. doi
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.
Publications
- T. Knöfel and N. Sträter. X-ray structure of the Escherichia coli periplasmic 5'-nucleotidase containing a dimetal catalytic site. Nature Struct. Biol. 1999, 6, 448-453.
- T. Knöfel and N. Sträter. Mechanism of hydrolysis of phosphate esters by the dimetal center of 5'-nucleotidase based on crystal structures. J. Mol. Biol. 2001, 309, 239-254.
- R. Schultz-Heienbrok, T. Maier and N. Sträter. A Large Hinge Bending Domain Rotation is Required for the Catalytic Function of E. coli 5'-Nucleotidase. Biochemistry 2005, 44, 2244-2252.
- K. Knapp, M. Zebisch, J. Pippel, A. El-Tayeb, C. E. Müller and N. Sträter. Crystal Structure of the Human Ecto-5'-Nucleotidase (CD73): Insights into the Regulation of Purinergic Signaling. Structure 2012, 20, 2161-2173.
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.
Biocatalysis
Proteins as catalysts are commonly referred to as enzymes. The term biocatalysis is usually used for the application of enzymes in biotechnological applications (e.g. enzymes in detergents or the food industry) or in organic chemical transformations (on a technical or even only on a laboratory scale).
We are interested in both the elucidation of the reaction mechanism of natural enzymes and their mode of action as well as in the engineering of enzymes for biocatalysis. For the former, we refer to the enzyme mechanisms of NTPDases and CD73 described on this page as examples. Two biocatalysis projects described in more detail below are the flavin-dependent monooxygenases for stereospecific organic transformations and enzymes for the environmentally friendly degradation of PET (polyethylene terephthalate) plastics, so-called PETases.
Polyethylene terephthalate (PET) is a widely used plastic, with the PET bottle certainly being the best-known product. The pollution of the environment by plastics is a challenge that is increasingly recognised. Solutions to this problem can lie in more environmentally friendly and effective degradation processes for recycling or to less polluting reaction products. One problem is the high stability of plastics like PET in the environment. Enzymes, on the other hand, are known for their enormous activity under environmentally compatible reaction conditions (ambient temperature, water as solvent). Indeed, there are enzymes in nature that can hydrolyse PET efficiently under mild conditions.
In 2014, we elucidated the first structure of a thermostable PET hydrolase in cooperation with Wolfgang Zimmermann's group in Leipzig in order to understand the structural basis of how this enzyme works. These enzymes are now a focus of worldwide research activities in biocatalysis. The aim is to further increase the effectiveness of the enzymes, using the screening of libraries of natural enzymes or genes, random mutagenesis and structure-based enzyme design as methods. We are also continuing our research on plastic-degrading enzymes in cooperation with Christian Sonnendecker and Wolfgang Zimmermann.
Structure-based drug development
The enzyme CD73 cleaves extracellular AMP to adenosine and phosphate and it is the main source of immunosuppressive adenosine. Many tumours overexpress the enzyme in order to escape the host immune response. Inhibition of CD73 by antibodies or synthetic small molecules has therefore been identified as a promising approach for cancer immunotherapy. Several antibodies and some organic molecules are currently in clinical trials.
We have accompanied the development of small molecule inhibitors of CD73 starting from the ADP analogue AMPCP (see also the figure above in the chapter on CD73 in extracellular signal transduction). The group of Christa Müller from the University of Bonn has developed derivatives of AMPCP in which a chlorine substituent at the C2 atom and a benzyl group at the amino group of the adenine increased the affinity from 88 nM for AMPCP to 0.32 nM for PSB12489. The compound AB680, developed by Arcus Biosciences, which was the first small molecule compound to enter clinical phases, inhibits CD73 even at 5 pM. The figure shows the binding mode of AB680 to CD73 as determined by us.
In current research, we are investigating the binding of further improved or alternative inhibitors, using, for example, pyrimidines as nucleobases instead of adenine.
Publications
- S. Bhattarai, J. Pippel, E. Scaletti, R. Idris, M. Freundlieb, G. Rolshoven, C. Renn, SY Lee, A. Abdelrahman, H. Zimmermann, A. El-Tayeb, C.E. Müller, N. Sträter.
2-Substituted α,β-Methylen -ADP Derivatives: Potent Competitive Ecto-5'-nucleotidase (CD73) Inhibitors with Variable Binding Modes.
J. Med Chem. 2020, 63 (6): 2941-2957. - S. Bhattarai, J. Pippel, A. Meyer, M. Freundlieb, C. Schmies, A. Abdelrahman et al.
X-Ray Co-Crystal Structure Guides the Way to Subnanomolar Competitive Ecto-5'-Nucleotidase (CD73) Inhibitors for Cancer Immunotherapy
Advanced Therapeutics 2019, 2 (10), 1900075.