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 & PRIME;-nucleotidase (h-ecto-5 & PRIME;-NT,CD73) isa homodimeric Zn2+-binding metallophosphoesterase thathydrolyzes adenosine 5 & PRIME;-monophosphate (5 & PRIME;-AMP) to adenosineand phosphate. h-Ecto-5 & PRIME;-NT is a key enzyme in purinergic signalingpathways and has been recognized as a promising biological targetfor several diseases, including cancer and inflammatory, infectious,and autoimmune diseases. Despite its importance as a biological target,little is known about h-ecto-5 & PRIME;-NT dynamics, which poses aconsiderable challenge to the design of inhibitors of this targetenzyme. Here, to explore h-ecto-5 & PRIME;-NT flexibility, all-atomunbiased molecular dynamics (MD) simulations were performed. Remarkabledifferences in the dynamics of the open (catalytically inactive) andclosed (catalytically active) conformations of the apo-h-ecto-5 & PRIME;-NTwere observed during the simulations, and the nucleotide analogueinhibitor AMPCP was shown to stabilize the protein structure in theclosed conformation. Our results suggest that the large and complexdomain motion that enables the h-ecto-5 & PRIME;-NT open/closed conformationalswitch is slow, and therefore, it could not be completely capturedwithin the time scale of our simulations. Nonetheless, we were ableto explore the faster dynamics of the h-ecto-5 & PRIME;-NT substratebinding site, which is mainly located at the C-terminal domain andwell 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 bindingsite. Finally, conformational states of the substrate binding sitewith higher druggability scores than the crystal structure were identified.In summary, our study provides valuable insights into h-ecto-5 & PRIME;-NTstructural flexibility, which can guide the structure-based designof novel h-ecto-5 & PRIME;-NT inhibitors.