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 Zn2+-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.