Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Thymidylate synthase (TS) is crucial for DNA synthesis, catalyzing the methylation of deoxyuridine monophosphate (dUMP).
• Understanding the structural basis of TS catalysis is essential for developing targeted therapies.

Purpose of the Study:

• To elucidate the structure of the ternary complex of Escherichia coli thymidylate synthase (TS) with dUMP and a methylenetetrahydrofolate analogue.
• To investigate the mechanism of dUMP methylation and the enzyme's conformational changes during catalysis.

Main Methods:

• X-ray crystallography was used to solve the structure of the TS-dUMP-cofactor analogue complex at 1.97-A resolution.
• Multiple isomorphous replacement and refinement techniques were employed.
• Comparison with the unliganded TS structure was performed.

Main Results:

• The ternary complex structure reveals that C-6 of dUMP covalently binds to Cys-198(146) during catalysis.
• Conserved residues form specific hydrogen bonds and hydrophobic interactions, sequestering reactants within the active site.
• A ligand-induced conformational change involves segmental accommodation of protein elements, closing the active site.
• A secondary binding site for the cofactor analogue was identified.

Conclusions:

• The determined structure provides direct insight into the mechanism of dUMP methylation by TS.
• Ligand binding induces significant conformational changes, creating a protected active site.
• The discovery of a secondary binding site suggests potential regulatory or alternative functional roles.