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 Center for Biotechnology and Biomedicine
 Institute of Bioanalytical Chemistry
  Structural analysis of biopolymers
  Prof. Dr. Norbert Sträter

Ecto-5'-Nucleotidase (CD73): Generation of extracellular adenosine from AMP

Ecto-5'-Nucleotidase (e5NT), also known as CD73, is part of the purinergic signaling cascade in that it hydrolyzes AMP to adenosine. Thereby, adenosine signaling via P1 receptors is switched on.
E5NT is involved in the development of chronic pain, hypoxia and inflammation. Furthermore, CD73 has been shown to be overexpressed in many cancer types for tumor promotion and metastasis. CD73 inhibitors are of interest for cancer immunotherapy.

We expressed e5NT in E. coli without the C-terminal GPI signal sequence which naturally anchors the enzyme to the cell membrane. The protein was refolded from inclusion bodies, purified and finally crystallized. E5NT shows a similar structure as its bacterial homolog: the two-domain enzyme contains the dimetal center in the N-terminal domain (blue and orange) and the substrate binding pocket in the C-terminal domain (green and yellow). However, e5NT adopts a dimeric arrangment via interaction of the C-terminal domains [3].
Furthermore, e5NT was crystallized in open and closed conformations which indicate domain rotations of up to 114°.
domain motion cd73

Domain motion of the dimer of e5NT

psb11552 inhibitor bound to cd73
Binding mode of PSB11552 to the substrate binding pocket of e5NT
E5NT is specific for the hydrolysis of AMP (whereas E. coli 5NT hydrolyzes ATP, ADP and AMP). From the current data we hypothesize that the domain motion controls the substrate specificity of e5NT.

Future work aims to further our understanding of the exclusive specificity of e5NT towards AMP which distinguishes the mammalian enzyme from the bacterial homologs.
Furthermore, due to its role in the development of cancer and other diseases, e5NT is an important target in structure based drug design. We aim to find lead structures to contribute to the development of pharmaceuticals.


Karen Knapp
Matthias Zebisch
Jan Pippel
Emma R. Scaletti


Professor Dr. Christa Müller, Universität Bonn, Pharmazeutisches Institut


  1. N. Sträter (2006).  Ecto-5'-Nucleotidase: Structure Function Relationships. Purinergic Signalling, 2, 343-350.
  2. K. Knapp, M. Zebisch, J. Pippel, A. El-Tayeb, C. E. Müller, N. Sträter (2012). Crystal Structure of the Human Ecto-5'-Nucleotidase (CD73): Insights into the Regulation of Purinergic Signaling. Structure, 20, 2161-2173.
  3. K. Knapp, M. Zebisch, N. Sträter (2012). Crystallization and preliminary X-ray analysis of the open form of human ecto-5'-nucleotidase. Acta Cryst. Sect. F, 68, 1545-1549.
  4. S. Bhattarai, M. Freundlieb, J. Pippel, A. Meyer, A. Abdelrahman, A. Fiene, S.Y. Lee, H. Zimmermann, G.G. Yegutkin, N. Sträter, A. El-Tayeb, C.E. Müller (2015). α,β-Methylene-ADP (AOPCP) Derivatives and Analogues: Development of Potent and Selective ecto-5'-Nucleotidase (CD73) Inhibitors. J. Med. Chem., 58, 6248-6263.