Effects of ligand binding and conformational switching on intracellular stability of human thymidylate synthase

Abstract

Thymidylate synthase (TS) is the target in colon cancer therapeutic protocols utilizing such drugs as 5-fluorouracil and raltitrexed. The effectiveness of these treatments is hampered by emerging drug resistance, usually related to increased levels of TS. Human TS (hTS) is unique among thymidylate synthases from all species examined as its loop 181–197 can assume two main conformations related by rotation of 180°. In one conformation, “active”, the catalytic Cys-195 is positioned in the active site; in the other conformation, “inactive”, it is at the subunit interface. Also, in the active conformation, region 107–128 has one well-defined conformation while in the inactive conformation this region assumes multiple conformations and is disordered in crystals. The native protein exists in apparent equilibrium between the two conformational states, while the enzyme liganded with TS inhibitors assumes the active conformation. The native protein has been reported to bind to several mRNAs, including its own mRNA, but upon ligation, RNA binding activity is lost. Ligation of TS by inhibitors also stabilizes it to turnover. Since currently used TS-directed drugs stabilize the active conformation and slow down the enzyme degradation, it is postulated that inhibitors of hTS stabilizing the inactive conformation of hTS should cause a down-regulation in enzyme levels as well as inactivate the enzyme.