Magic-Angle Spinning Solid-State NMR Spectroscopy of Nanodisc-Embedded Human CYP3A4

Abstract

Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not been revealed so far because X-ray crystallography studies are available only for the soluble domain of CYP3A4. Here we report sample preparation and initial magic-angle spinning (MAS) solid-state NMR (SSNMR) of CYP3A4 (Delta3-12) embedded in a nanoscale membrane bilayer, or Nanodisc. The growth protocol yields approximately 2.5 mg of the enzymatically active, uniformly 13C,15N-enriched CYP3A4 from 1 L of growth medium. Polyethylene glycol 3350-precipitated CYP3A4 in Nanodiscs yields spectra of high resolution and sensitivity, consistent with a folded, homogeneous protein. CYP3A4 in Nanodiscs remains enzymatically active throughout the precipitation protocol as monitored by bromocriptine binding. The 13C line widths measured from 13C-13C 2D chemical shift correlation spectra are approximately 0.5 ppm. The secondary structure distribution within several amino acid types determined from 13C chemical shifts is consistent with the ligand-free X-ray structures. These results demonstrate that MAS SSNMR can be performed on Nanodisc-embedded membrane proteins in a folded, active state. The combination of SSNMR and Nanodisc methodologies opens up new possibilities for obtaining structural information on CYP3A4 and other integral membrane proteins with full retention of functionality.

Conformational Plasticity and Structure/Function Relationships in Cytochromes P450

“…While the de novo determination of a cytochrome P450 structure by NMR methods has not been reported, it has been shown that extensive sequential resonance assignments (a prerequisite for structure determination) can be made for soluble P450s (5), and high-quality solid state NMR spectra have been demonstrated for membrane-associated mammalian P450s as well (51). NMR methods often require a significant investment of time and resources to reach the point at which interpretable atomic resolution information concerning structure and dynamics of P450 can be obtained: Isotopically labeled samples must be prepared of sufficient concentration and purity for spectroscopy, a variety of multidimensional NMR experiments performed, and then sequential assignments must be made.…”

“…However, combined with selective and uniform isotopic labeling, recent developments in solid-state NMR methods promise to make eukaryotic P450s more accessible (51,124). Using solid state NMR, the McDermott group has shown that substrate binding and spin state in CYP102 is temperature dependent, suggesting substrate reorientation as a function of temperature in that enzyme (48,49).…”

Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles

“…Unlike other systems such as liposomes or detergent micelles, nanodiscs are relatively monodisperse (∼100 Å in diameter) and stable. Furthermore, protein-nanodisc complexes have proven to be easy to construct, are readily purified and concentrated, and are applicable to single particle structural analysis (5). We have now succeeded in assembling PA-pore lipidnanodisc and PA-pore:vesicle complexes and have reconstructed the nanodisc/vesicle-inserted PA pore by using EM of negatively stained specimens.…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…In particular, solution NMR can report not only on protein tertiary structure, but also on conformational dynamics which are important to the interaction with protein partners (Lampe et al, 2008(Lampe et al, , 2010). An additional advantage is that protein NMR can be performed in the presence of lipid bilayer mimetics, such as bicells (Ahuja et al, 2013) or nanodiscs (Kijac et al, 2007;Gluck et al, 2009), allowing the investigator to examine the effect of lipid on protein-protein interactions. Protein NMR has been most widely used to examine interactions between CYP enzymes and their electron transfer partners (Ahuja et al, 2013;Estrada et al, 2013Estrada et al, , 2014Estrada et al, , 2016.…”