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Engineering dimer mutants of human geranylgeranyl pyrophosphate synthase
Sean J Ezekiel1, Mackenzie Searle1, Jaeok Park1
1Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada.
Plos One
|January 15, 2025
Summary
Researchers engineered dimeric forms of geranylgeranyl pyrophosphate synthase (GGPPS) to overcome structural challenges for cancer therapy development. These stable mutants retain activity and facilitate detailed structural analysis of ligand binding.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Geranylgeranyl pyrophosphate synthase (GGPPS) is crucial for protein prenylation and cellular signaling, making it a key target for cancer therapies.
- The native hexameric structure of GGPPS complicates high-resolution crystallographic studies.
- Developing GGPPS inhibitors requires detailed understanding of its ligand-binding interactions.
Purpose of the Study:
- To engineer dimeric variants of human GGPPS to enable high-resolution crystallographic analysis.
- To investigate the structural and functional properties of GGPPS dimers.
- To facilitate the rational design of novel GGPPS-targeting therapeutic agents.
Main Methods:
- Site-directed mutagenesis was employed to disrupt inter-dimer interactions and generate stable dimeric GGPPS mutants.
- Enzyme activity assays were performed to confirm retained catalytic function of the mutants.
- Differential scanning fluorimetry (DSF) assessed protein stability and inhibitor binding.
- X-ray crystallography was used to determine the high-resolution structure of a GGPPS dimer mutant.
Main Results:
- Three stable dimeric GGPPS mutants (Y246D/C247L, Y246D/C205A, Y246K/C247L) were successfully generated.
- All mutants exhibited wild-type catalytic activity.
- The Y246D/C247L mutant yielded a 2.1 Å crystal structure, revealing isopentenyl pyrophosphate binding and unexpected intermolecular disulfide bonds.
- Mutants showed similar thermal stability and inhibitor responses compared to wild-type GGPPS.
Conclusions:
- Engineered dimeric GGPPS mutants are viable alternatives to the wild-type enzyme for structural studies.
- The dimeric mutants provide enhanced crystallizability, facilitating detailed structural insights.
- These findings support the use of dimeric GGPPS mutants as molecular tools for drug discovery targeting GGPPS.
