Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in...

Area of Science:

• Biochemistry
• Structural Biology
• Drug Discovery

Background:

• Human thymidylate synthase (hTS) is an essential enzyme and a validated target for antiproliferative drugs.
• hTS functions as an obligate homodimer, making its protein-protein interactions crucial for activity.

Purpose of the Study:

• To identify specific residues at the hTS monomer-monomer interface that can be mutated to perturb intersubunit interactions.
• To explore the potential of these mutations in designing novel inhibitors targeting the dimer interface.

Main Methods:

• Computational identification of putative hotspot residues at the hTS dimer interface.
• Site-directed mutagenesis to create single-point alanine mutants at identified interface residues.
• Förster Resonance Energy Transfer (FRET)-based assay to measure dimer dissociation constants.
• Enzyme activity assays to assess the impact of mutations on catalytic function and substrate affinity (KM).

Main Results:

• Single-point mutations at the monomer-monomer interface significantly reduced dimer stability, with dissociation constants increasing up to ~1 mM compared to wild-type hTS (~60 nM).
• Computational predictions of mutation effects correlated well with experimental FRET data.
• Mutations remote from the active site generally retained enzyme activity, though often with increased substrate KM values.
• The reduced dimer stability in mutants was observed to potentially facilitate small molecule access to the dimer interface.

Conclusions:

• Mutations at the hTS dimer interface can effectively destabilize the homodimer while largely preserving enzymatic activity.
• Destabilizing the hTS dimer through interface mutations offers a promising strategy for developing new inhibitors that target protein-protein interactions.
• This approach could lead to novel antiproliferative drugs with unique mechanisms of action.