Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells

J Biomol Struct Dyn. 2017 May;35(7):1474-1490. doi: 10.1080/07391102.2016.1186560. Epub 2016 Aug 5.


Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation in vivo, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an in silico model of human DHFR-TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.

Keywords: molecular dynamics; protein–protein complex model; protein–protein interaction; protein–protein interface; thymidylate synthesis.

MeSH terms

  • Antimetabolites, Antineoplastic / chemistry*
  • Antimetabolites, Antineoplastic / pharmacology
  • Binding Sites
  • Cell Cycle / drug effects
  • Cell Cycle / genetics
  • Cell Line
  • Crystallography, X-Ray
  • DNA / chemistry*
  • DNA / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / enzymology*
  • Fluorouracil / chemistry*
  • Fluorouracil / pharmacology
  • HCT116 Cells
  • Humans
  • Kinetics
  • Methotrexate / chemistry*
  • Methotrexate / pharmacology
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Multienzyme Complexes / antagonists & inhibitors
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / metabolism
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Substrate Specificity
  • Tetrahydrofolate Dehydrogenase / chemistry*
  • Tetrahydrofolate Dehydrogenase / genetics
  • Tetrahydrofolate Dehydrogenase / metabolism
  • Thermodynamics
  • Thymidylate Synthase / antagonists & inhibitors
  • Thymidylate Synthase / chemistry*
  • Thymidylate Synthase / genetics
  • Thymidylate Synthase / metabolism


  • Antimetabolites, Antineoplastic
  • Multienzyme Complexes
  • thymidylate synthase-dihydrofolate reductase
  • DNA
  • Tetrahydrofolate Dehydrogenase
  • Thymidylate Synthase
  • Fluorouracil
  • Methotrexate