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Updated: Jun 5, 2026
Modeling Ligands into Maps Derived from Electron Cryomicroscopy
Published on: July 19, 2024
Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding
Lydia M Gibson1, Lesa R Celeste, Leslie L Lovelace
1Department of Chemistry and Biochemistry, University of South Carolina, Columbia, 29208, USA.
Abstract:
Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed that hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.
Insights
A new crystal form of human thymidylate synthase (hTS) enables drug discovery. This structure reveals how hTS binds cancer drug precursors and glutathione, aiding in developing new cancer therapies.
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- Thymidylate synthase (TS) is a crucial enzyme in DNA synthesis and a validated target for cancer chemotherapy.
- Understanding the structural basis of TS inhibition is key to developing effective anti-cancer drugs.
Purpose of the Study:
- To characterize a novel crystal form of the R163K variant of human thymidylate synthase (hTS).
- To utilize this crystal form for structural studies of ligand binding within the active site.
- To facilitate high-throughput screening of potential drug candidates targeting hTS.
Main Methods:
- Crystallization of the R163K variant of human thymidylate synthase.
- X-ray crystallography to determine the structure of hTS-ligand complexes.
- Crystal soaking experiments with FdUMP, dUMP, and glutathione analogs.
Main Results:
- A new crystal form of hTS with five subunits per asymmetric unit was obtained, featuring loop 181-197 in an active conformation.
- Crystal structures of hTS complexed with FdUMP and dUMP were determined, validating the crystal form for drug candidate analysis.
- Binding of oxidized glutathione to hTS was observed, with two distinct asymmetric binding modes: covalent modification of Cys195 and non-covalent adduct formation with reduced glutathione.
Conclusions:
- The newly characterized crystal form of hTS is suitable for high-throughput screening of potential anti-cancer drugs.
- Structural insights into the binding of FdUMP, dUMP, and glutathione provide a foundation for rational drug design.
- The asymmetric binding of glutathione suggests complex regulatory mechanisms or potential drug interaction pathways involving TS.
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