The active–inactive transition of human thymidylate synthase: Targeted molecular dynamics simulations

Abstract

Human thymidylate synthase (hTS) is an established anticancer target. It catalyses the production of 2'-deoxythymidine-5'-monophosphate, an essential building block for DNA synthesis. Because of the development of cellular drug resistance against current hTS inhibitors, alternative inhibition strategies are needed. hTS exists in two forms, active and inactive, defined by the conformation of the active-site (AS) loop, which carries the catalytic cysteine, C195. To investigate the mechanism of activation and inactivation, targeted molecular dynamics (TMD) simulations of the transitions between active and inactive states of hTS were performed. Analysis of changes in the dihedral angles in the AS loop during different TMD simulations revealed complex conformational transitions. Despite hTS being a homodimeric enzyme and the conformational transition significantly involving the dimer interface, the transition occurs in an asymmetric, sequential manner via an ensemble of pathways. In addition to C195, which reoriented during the simulations, other key residues in the rotation of the AS loop included W182 and R185. The interactions of the cognate bulky W182 residues at the dimer interface hindered the simultaneous twist of the AS loops in the hTS dimer. Interactions of R185, which is unique for hTS, with ligands at different allosteric sites affected the activation transition. In addition to providing insights into the activation/inactivation mechanism of hTS and how conformational transitions can occur in homodimeric proteins, our observations suggest that blocking of AS loop rotation by ligands binding in the large cavity between the loops could be one way to stabilize inactive hTS and inhibit the enzyme.

Hotspots in an Obligate Homodimeric Anticancer Target. Structural and Functional Effects of Interfacial Mutations in Human Thymidylate Synthase

“…In simulations of the active/inactive transition of hTS, we found that the conformational change is hindered by the two W182 side chains which must avoid steric clashes as they reorient. 38 The W182A mutation introduces space at the dimer interface region, and this, besides relaxing the steric hindrance to the active/inactive transformation, likely favors the more extended inactive conformation of the active site loop.…”

Three pairs of weak interactions precisely regulate the G-loop gate of Kir2.1 channel

“… where the force constant is represented by k , N is the number of atoms which are set as target structure, RMSD(t) between the current coordinates and the target structure is computed (after first superimposing the target structure and the initial coordinates), and RMSD*(t) evolves linearly from the initial RMSD at the first Targeted MD step to the final RMSD at the last Targeted MD step. RMSD*(t) tends to zero is the criterion to end the Targeted MD …”

Downregulation of thymidylate synthase with arsenic trioxide in lung adenocarcinoma

“…Intriguingly, the inactive human TYMS binds to TYMS mRNA, thus inhibiting TYMS protein synthesis (34). When activated, the active site loop region (residues 181-197) of TYMS is rotated about 180˚, so that the catalytic cysteine (C195) is then exposed and oriented towards the dimer interface for its function (35). Upon treatment with ATO, TYMS activity was reduced.…”