Structure of human thymidylate synthase under low-salt conditions

Human thymidylate synthase, a target in cancer chemotherapy, was crystallized from PEG 3350 with 30 mM ammonium sulfate (AS) in the crystallization medium. The crystals are isomorphous with the high-salt crystals (∼2.0 M AS) and the structure has been solved and refined (R = 22.6%, R_free = 24.3%) at 1.8 Å resolution. The high- and low-AS-concentration structures are quite similar, with loop 181–197 is in the inactive conformation. Also, residues 95–106 and 129–135 (eukaryotic inserts region) show high mobility as assessed by poor electron density and high values of crystallographic temperature factors (residues 1–25 and 108–129 are disordered in both structures). The high mobility of this region may reflect the situation at physiological ionic strength. Of the four sulfate ions observed bound at 2.0 M AS, only two are present at 30 mM AS. The inactive conformation appears to be stabilized by the side chain of Val3 or a leucine residue from the disordered regions. The low-salt conditions of these crystals should be much more suitable for the study of thymidylate synthase inhibitors, especially those that utilize sulfate-binding sites to stabilize the inactive conformation of loop 181–197.