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Crystal structures of rat thymidylate synthase inhibited by Tomudex, a potent anticancer drug
R R Sotelo-Mundo1, J Ciesla, J M Dzik
1Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Abstract:
Two crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex (ZD1694) have been determined to resolutions of 3.3 and 2.6 A. Tomudex is one of several new antifolates targeted to TS and the first to be approved for clinical use. The structures represent the first views of any mammalian TS bound to ligands and suggest that the rat protein undergoes a ligand-induced conformational change similar to that of the Escherichia coli protein. Surprisingly, Tomudex does not induce the "closed" conformation in rat TS that is seen on binding to E. coli TS, resulting in inhibitor atoms that differ in position by more than 1.5 A. Several species-specific differences in sequence may be the reason for this. Phe 74 shifts to a new position in the rat complex and is in van der Waals contact with the inhibitor, while in the E. coli protein the equivalent amino acid (His 51) hydrogen bonds to the glutamate portion of the inhibitor. Amino acids Arg 101, Asn 106, and Met 305 make no contacts with the inhibitor in the open conformation, unlike the equivalent residues in the E. coli protein (Thr 78, Trp 83, and Val 262). dUMP binding is similar in both proteins, except that there is no covalent adduct to the active site cysteine (Cys 189) in the rat structures. Two insertions in the rat protein are clearly seen, but the N-termini (residues 1-20) and C-termini (residues 301-307) are disordered in both crystal forms.
Insights
Crystal structures reveal rat thymidylate synthase (TS) binding the anticancer drug Tomudex differently than E. coli TS. This difference in ligand-induced conformational change may explain species-specific drug interactions.
Area of Science:
- Biochemistry
- Structural Biology
- Pharmacology
Background:
- Thymidylate synthase (TS) is a critical enzyme in DNA synthesis and a target for anticancer drugs.
- Antifolates like Tomudex are designed to inhibit TS, but their precise interactions with mammalian TS are not fully understood.
- Previous studies focused on bacterial TS, leaving mammalian TS-ligand complexes largely uncharacterized.
Purpose of the Study:
- To determine the crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex.
- To elucidate the structural basis for Tomudex binding to mammalian TS.
- To compare the ligand-induced conformational changes in rat TS with those observed in bacterial TS.
Main Methods:
- X-ray crystallography was used to determine two crystal structures of rat TS complexed with dUMP and Tomudex.
- High-resolution structural data (2.6 and 3.3 A) were obtained and analyzed.
- Comparative structural analysis was performed between rat and Escherichia coli TS structures.
Main Results:
- The crystal structures reveal distinct binding modes of Tomudex in rat TS compared to E. coli TS.
- Tomudex does not induce the same "closed" conformation in rat TS as observed in E. coli TS, with key inhibitor atoms differing in position.
- Species-specific amino acid differences, such as Phe 74 in rat TS, influence inhibitor interactions.
- dUMP binding is generally conserved, but the active site cysteine (Cys 189) does not form a covalent adduct in the rat structures.
- Disordered N- and C-termini were observed in both crystal forms.
Conclusions:
- Rat thymidylate synthase undergoes a ligand-induced conformational change, but it differs from the response of E. coli TS to Tomudex.
- Species-specific sequence variations in TS contribute to differential drug binding and conformational responses.
- These findings provide insights into the structural basis for antifolate drug action in mammalian systems and may inform future drug design.