Working at the membrane interface: Ligand‐induced changes in dynamic conformation and oligomeric structure in human aromatase

Nardo, Giovanna Di; Cimicata, Giuseppe; Baravalle, Roberta; Dell'Angelo, V.; Ciaramella, Alberto; Catucci, Gianluca; Ugliengo, Piero; Gilardi, Gianfranco

doi:10.1002/bab.1613

Cited by 18 publications

(17 citation statements)

References 51 publications

(70 reference statements)

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“…In addition to function in a soluble form, CYPs are able to attach to membrane with its N-terminal, and interact with its redox partner CYP reductase in the membrane environment, which are essential for electron transfer in the catalytic cycle 51 . Interactions with membrane have been observed to modulate the conformational dynamics of human aromatase (CYP19A1) and alter the substrate/inhibitor access channels 52 . Computational simulations and experimental data have also revealed that interactions of CYP enzymes with partner reductase in the membrane could affect the substrate binding 53,54 .…”

Section: Discussionmentioning

confidence: 99%

Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

Chen

2020

Sci Rep

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Cytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

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Section: Discussionmentioning

confidence: 99%

Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

Chen

2020

Sci Rep

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“…CYP19A1 activity is also inhibited by bisphenol A and sildenafil [55]. CYP19A1 inhibitors like anastrozole are used in the treatment of breast cancer and to treat the excess production of estrogen in men [57].…”

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confidence: 99%

Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H

Parween

Nardo

Baj

et al. 2020

The Journal of Steroid Biochemistry and Molecular Biology

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Aromatase (CYP19A1) converts androgens into estrogens and is required for female sexual development and growth and development in both sexes. CYP19A1 is a member of cytochrome P450 family of heme-thiolate monooxygenases located in the endoplasmic reticulum and depends on reducing equivalents from the reduced nicotinamide adenine dinucleotide phosphate via the cytochrome P450 oxidoreductase coded by POR. Both the CYP19A1 and POR genes are highly polymorphic, and mutations in both these genes are linked to disorders of steroid biosynthesis. We have previously shown that R264C and R264H mutations in CYP19A1, as well as mutations in POR, result in a reduction of CYP19A1 activity. The R264C is a common polymorphic variant of CYP19A1, with high frequency in Asian and African populations. Polymorphic alleles of POR are found in all populations studied so far and, therefore, may influence activities of CYP19A1 allelic variants. So far, effects of variations in POR on enzymatic activities of allelic variants of CYP19A1 or any other steroid metabolizing cytochrome P450 proteins have not been studied. Here we are reporting the effects of three POR variants on the aromatase activities of two CYP19A1 variants, R264C and R264H. We used bacterially expressed and purified preparations of WT and variant forms of CYP19A1 and POR and constructed liposomes with embedded CYP19A1 and POR proteins and assayed the CYP19A1 activities using radiolabeled androstenedione as a substrate. With the WT-POR as a redox partner, the R264C-CYP19A1 showed only 15% of aromatase activity, but the R264H had 87% of aromatase activity compared to WT-CYP19A1. With P284L-POR as a redox partner, R264C-CYP19A1 lost all activity but retained 6.7% of activity when P284T-POR was used as a redox partner. The R264H-CYP19A1 showed low activities with both the POR-P284L as well as the POR-P284T. When the POR-Y607C was used as a redox partner, the R264C-CYP19A1 retained around 5% of CYP19A1 activity. Remarkably, The R264H-CYP19A1 had more than threefold higher activity compared to WT-CYP19A1 when the POR-Y607C was used as the redox partner, pointing towards a beneficial effect. The slight increase in activity of R264C-CYP19A1 with the P284T-POR and the threefold increase in activity of the R264H-CYP19A1 with the Y607C-POR point towards a conformational effect and role of protein-protein interaction governed by the R264C and R264H substitutions in the CYP19A1 as well as P284L, P284T and Y607C variants of POR. These studies demonstrate that the allelic variants of P450 when present with a variant form of POR may show different activities, and combined effects of variations in both the P450 enzymes as well as in the POR should be considered when genetic data are available. Recent trends in the whole-exome and whole-genome sequencing as diagnostic tools will permit combined evaluation of variations in multiple genes that are interdependent and may guide treatment options by adjusting therapeutic interventions based on laboratory analysis.

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“…A topological characterization of the aromatase structure has been attempted using the available crystallographic model of substrate-containing aromatase (PDB code 4kq8), and an in silico version of the ligand-free protein, obtained through a molecular dynamics simulation [25]. A contact network approach consists of a coarse-graining algorithm that reduces the amino acids of a protein into "nodes" of a tridimensional grid, identifying the position of each residue with that of its α-carbon.…”

Section: Contact Network Analysismentioning

confidence: 99%

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

Nardo

Venere

Zhang

et al. 2021

Preprint

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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.

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Working at the membrane interface: Ligand‐induced changes in dynamic conformation and oligomeric structure in human aromatase

Cited by 18 publications

References 51 publications

Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H

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

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