Designing novel adenine clamp-CD73 mutants delineating surface expression and enzyme activity through in silico and in vitro engineering

NT5E, a 5' Ectonucleotidase, is an emerging hotspot for cancer immunotherapies. However, available enzymatic inhibitors have shown limited promise, indicating activity-independent roles. Delineating activity-dependent and independent roles of this protein with precision requisites identification of critical residues impacting activity without hampering surface expression/stability. Conserved Phenylalanine (F) at 417 and 500 positions, which form a π stack with substrate-AMP, was chosen as the region of interest for in silico mutagenesis. F417 and F500 were mutated to Valine (V) individually using Missense3D to obviate broad structural damage. CD73-F417V/F500V dual mutant was then subjected to Molecular Dynamics simulations for prediction of stability and structural variations in the protein over time in comparison to wild-type crystal structure. Gibbs Free energy indicated a significant decrease in ligand binding affinity of CD73-F417V/F500V. RMSD and RMSF analysis of MD trajectories indicated increased mobility, suggesting weaker ligand binding affinity within the catalytic site of the mutant potentially impacting functional dynamics and protein-ligand contacts. Overexpression of CD73-F417V/F500V mutant in CD73 null-HEKs indicated stable surface expression but complete loss of Ecto-nucleotidase activity, thus providing a tool to dissect activity-dependent and independent functions of NT5E.