Volume 257, August 2024, 112579Author links open overlay panel, , , Highlights•

Aromatase nanodiscs were characterized by small angle x-ray and neutron scattering (SAXS/SANS).

•

A hybrid structural model of an aromatase nanodisc was derived from SAXS and SANS.

•

The orientation consistent with a substrate recognition at the membrane interface.

Abstract

Human aromatase (CYP19A1), the cytochrome P450 enzyme responsible for conversion of androgens to estrogens, was incorporated into lipoprotein nanodiscs (NDs) and interrogated by small angle X-ray and neutron scattering (SAXS/SANS). CYP19A1 was associated with the surface and centered at the edge of the long axis of the ND membrane. In the absence of the N-terminal anchor, the amphipathic A'- and G'-helices were predominately buried in the lipid head groups, with the possibly that their hydrophobic side chains protrude into the hydrophobic, aliphatic tails. The prediction is like that for CYP3A4 based on SAXS employing a similar modeling approach. The orientation of CYP19A1 in a ND is consistent with our previous predictions based on molecular dynamics simulations and lends additional credibility to the notion that CYP19A1 captures substrates from the membrane.

Section snippetsSmall-Angle X-Ray Scattering

SAXS data were collected at the SIBYLS beamline (12.3.1) at the Advanced Light Source, part of the Lawrence Berkeley National Laboratory. [[28], [29], [30]] Samples were shipped on ice overnight in 96-well plates. Triplicate sets of 30 μL ND samples were bracketed by two measurements of ND-free buffer (0.02 mol/L (M) Tris [pH 7.4], 0.1 M NaCl, 0.5 mM EDTA, 5 mM 2-mercaptoethanol) to reduce error in subtraction. To ensure the reference buffers were matched to those of the corresponding ND

Notes

Certain commercial materials, suppliers, and software are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. This manuscript has been coauthored by UT-Battelle, L.L.C., under contract no. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States

Funding

This work was supported by National Institutes of Health grant R01GM135414 awarded to J. C. H. Neutron scattering research conducted using the Bio-SANS instrument, a DOE Office of Science, Office of Biological and Environmental Research resource (FWP ERKP291), used resources at the High-Flux Isotope Reactor, a DOE Office of Science, Scientific User Facility operated by the Oak Ridge National Laboratory. This work was also conducted at the Advanced Light Source (ALS), a national user facility

CRediT authorship contribution statement

John C. Hackett: Writing – review & editing, Writing – original draft, Visualization, Investigation, Funding acquisition, Formal analysis, Conceptualization. Susan Krueger: Writing – review & editing, Formal analysis, Conceptualization. Volker S. Urban: Investigation, Formal analysis. Francisco Zárate-Pérez: Resources.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

John C Hackett reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors grateful to Martin Cramer Pedersen (Niels Bohr Institute, University of Copenhagen) for guidance on use of the WillItFit software.

References (33)N. Skar-Gislinge et al.Small-angle scattering from phospholipid nanodiscs: derivation and refinement of a molecular constrained analytical model form factor

Phys. Chem. Chem. Phys.

(2011)

S.A. Kynde et al.Small-angle scattering gives direct structural information about a membrane protein inside a lipid environment

Acta Crystallogr. D Biol. Crystallogr.

(2014)

M.C. Pedersen et al.WillItFit: a framework for fitting of constrained models to small-angle scattering data

J. Appl. Crystallogr.

(2013)

N. Skar-Gislinge et al.Small-angle scattering determination of the shape and localization of human cytochrome P450 embedded in a phospholipid nanodisc environment

Acta Crystallogr. D Biol. Crystallogr.

(2015)

R.W. Brueggemeier et al.Aromatase inhibitors in the treatment of breast cancer

Endocr. Rev.

(2005)

T.H. Bayburt et al.Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins

Nano Lett.

(2002)

View full text

© 2024 Elsevier Inc. All rights reserved.