Molecular Dynamics Simulations of the Human Ecto-5'-Nucleotidase (h-ecto-5'-NT, CD73): Insights into Protein Flexibility and Binding Site Dynamics.

Human ecto-5'-nucleotidase (h-ecto-5'-NT, CD73) is a homodimeric Zn-binding metallophosphoesterase that hydrolyzes adenosine 5'-monophosphate (5'-AMP) to adenosine and phosphate. h-Ecto-5'-NT is a key enzyme in purinergic signaling pathways and has been recognized as a promising biological target for several diseases, including cancer and inflammatory, infectious, and autoimmune diseases. Despite its importance as a biological target, little is known about h-ecto-5'-NT dynamics, which poses a considerable challenge to the design of inhibitors of this target enzyme. Here, to explore h-ecto-5'-NT flexibility, all-atom unbiased molecular dynamics (MD) simulations were performed. Remarkable differences in the dynamics of the open (catalytically inactive) and closed (catalytically active) conformations of the apo-h-ecto-5'-NT were observed during the simulations, and the nucleotide analogue inhibitor AMPCP was shown to stabilize the protein structure in the closed conformation. Our results suggest that the large and complex domain motion that enables the h-ecto-5'-NT open/closed conformational switch is slow, and therefore, it could not be completely captured within the time scale of our simulations. Nonetheless, we were able to explore the faster dynamics of the h-ecto-5'-NT substrate binding site, which is mainly located at the C-terminal domain and well conserved among the protein's open and closed conformations. Using the TRAPP ("Transient Pockets in Proteins") approach, we identified transient subpockets close to the substrate binding site. Finally, conformational states of the substrate binding site with higher druggability scores than the crystal structure were identified. In summary, our study provides valuable insights into h-ecto-5'-NT structural flexibility, which can guide the structure-based design of novel h-ecto-5'-NT inhibitors.