Polymorphism on human aromatase affects protein dynamics and substrate binding: spectroscopic evidence

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Human aromatase, a cytochrome P450 enzyme, is crucial for estrogen biosynthesis and a target for breast cancer therapy.
• Single-nucleotide polymorphisms (SNPs) like R264C and R264H can alter aromatase activity and disease risk.

Purpose of the Study:

• Investigate the impact of R264C and R264H mutations on human aromatase protein dynamics.
• Elucidate how these mutations affect ligand binding, substrate interaction, and catalytic function.

Main Methods:

• UV/FTIR spectroscopy to measure H/D exchange rates.
• Time-resolved fluorescence spectroscopy to analyze protein dynamics.
• Contact network analysis to map protein structural connectivity.

Main Results:

• Wild-type aromatase shows decreased H/D exchange rates upon ligand binding, indicating a more compact structure.
• Mutant aromatase variants exhibit similar H/D exchange rates in ligand-free and bound states, suggesting a lack of conformational change.
• Ligand binding fails to quench tryptophan-224 fluorescence in mutants, indicating compromised substrate binding/retention in the active site.
• Contact network analysis reveals altered connectivity, particularly in helix-G, where mutations occur.

Conclusions:

• Aromatase surface SNPs (R264C/R264H) reduce protein flexibility essential for substrate binding and catalysis.
• The observed effects suggest that helix G is a key region influencing aromatase dynamics and function.
• These findings provide a rationale for targeting helix G for aromatase inhibition in therapeutic strategies.