Computational study of the effects of protein tyrosine nitrations on the catalytic activity of human thymidylate synthase.

Tyrosine nitration is a widespread post-translational modification capable of affecting both the function and structure of the host protein molecule. Enzyme thymidylate synthase (TS), a homodimer, is a molecular target for anticancer therapy. Recently purified TS preparations, isolated from mammalian tissues, were found to be nitrated, suggesting this modification to appear endogenously in normal and tumor tissues. Moreover, human TS (hTS) nitration in vitro led to a by twofold lowered catalytic activity following nitration in average of 1 tyrosine residue per monomer (Dąbrowska-Maś et al. in Org Biomol Chem 10:323-331, 2012), with the modification identified by mass spectrometry at seven different sites (Y33, Y65, Y135, Y213, Y230, Y258 and Y301). In the present paper, combined computational approach, including molecular and essential dynamics and free energy computations, was used to predict the influence on the activity of hTS of nitration of each of the seven tyrosine residues. The simulations were based on the crystal structure of hTS ternary complex with dUMP and Tomudex (PDB code: 1I00), with the Tomudex molecule replaced by the molecule of TS cofactor analogue, tetrahydrofolate. The present results indicate that while with nitration of five out of seven residues (Y33, Y135, Y230, Y258 and Y301), single residue modification appears to have a strong reducing effect on the activity, with the remaining two, Y65 and Y213, no or a weaker influence is apparent. Taken together, these results demonstrate that tyrosine nitrations in the hTS enzyme show clear tendency to influence the structure and dynamics and, in turn, catalytic properties of the host enzyme. These effects are overall distance-dependent.