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

• Main Authors: Castrignanò, S. (Author), Di Nardo, G. (Author), Di Paola, L. (Author), Di Venere, A. (Author), Gilardi, G. (Author), Mei, G. (Author), Nicolai, E. (Author), Zhang, C. (Author)
• Format: Article
• Language: English
• Published: BioMed Central Ltd 2021
• Subjects: aromatase; Aromatase; Aromatase polymorphism; catalysis; Catalysis; Catalytic Domain; CYP19A1 protein, human; enzyme active site; Fluorescence; genetic polymorphism; genetics; human; Humans; Ligand binding; metabolism; Molecular modeling; Polymorphism, Genetic; protein binding; Protein Binding; spectrofluorometry; Spectrometry, Fluorescence
• Online Access: View Fulltext in Publisher

 Human aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy. Two single-nucleotide polymorphisms (R264C and R264H) have been shown to alter aromatase activity and they have been associated to an increased or decreased risk for estrogen-dependent pathologies. Here, the effect of these mutations on the protein dynamics is investigated by UV/FTIR and time resolved fluorescence spectroscopy. H/D exchange rates were measured by FTIR for the three proteins in the ligand-free, substrate- and inhibitor-bound forms and the data indicate that the wild-type enzyme undergoes a conformational change leading to a more compact tertiary structure upon substrate or inhibitor binding. Indeed, the H/D exchange rates are decreased when a ligand is present. In the variants, the exchange rates in the ligand-free and –bound forms are similar, indicating that a structural change is lacking, despite the single amino acid substitution is located in the peripheral shell of the protein molecule. Moreover, the fluorescence lifetimes data show that the quenching effect on tryptophan-224 observed upon ligand binding in the wild-type, is absent in both variants. Since this residue is located in the catalytic pocket, these findings suggest that substrate entrance and/or retention in the active site is partially compromised in both mutants. A contact network analysis demonstrates that the protein structure is organized in two main clusters, whose connectivity is altered by ligand binding, especially in correspondence of helix-G, where the amino acid substitutions occur. Our findings demonstrate that SNPs resulting in mutations on aromatase surface modify the protein flexibility that is required for substrate binding and catalysis. The cluster analysis provides a rationale for such effect, suggesting helix G as a possible target for aromatase inhibition. © 2021, The Author(s).