Cytochrome P450 3A4 (CYP3A4) is the
most abundant membrane-associated
isoform of the P450 family in humans and is responsible for biotransformation
of more than 50% of drugs metabolized in the body. Despite the large
number of crystallographic structures available for CYP3A4, no structural
information for its membrane-bound state at an atomic level is available.
In order to characterize binding, depth of insertion, membrane orientation,
and lipid interactions of CYP3A4, we have employed a combined experimental
and simulation approach in this study. Taking advantage of a novel
membrane representation, highly mobile membrane mimetic (HMMM), with
enhanced lipid mobility and dynamics, we have been able to capture
spontaneous binding and insertion of the globular domain of the enzyme
into the membrane in multiple independent, unbiased simulations. Despite
different initial orientations and positions of the protein in solution,
all the simulations converged into the same membrane-bound configuration
with regard to both the depth of membrane insertion and the orientation
of the enzyme on the surface of the membrane. In tandem, linear dichroism
measurements performed on CYP3A4 bound to Nanodisc membranes were
used to characterize the orientation of the enzyme in its membrane-bound
form experimentally. The heme tilt angles measured experimentally
are in close agreement with those calculated for the membrane-bound
structures resulted from the simulations, thereby verifying the validity
of the developed model. Membrane binding of the globular domain in
CYP3A4, which appears to be independent of the presence of the transmembrane
helix of the full-length enzyme, significantly reshapes the protein
at the membrane interface, causing conformational changes relevant
to access tunnels leading to the active site of the enzyme.
Membrane proteins reconstituted into phospholipid nanodiscs comprise a soluble entity accessible to solution small-angle X-ray scattering (SAXS) studies. It is demonstrated that using SAXS data it is possible to determine both the shape and localization of the membrane protein cytochrome P450 3A4 (CYP3A4) while it is embedded in the phospholipid bilayer of a nanodisc. In order to accomplish this, a hybrid approach to analysis of small-angle scattering data was developed which combines an analytical approach to describe the multi-contrast nanodisc with a free-form bead-model description of the embedded protein. The protein shape is then reconstructed ab initio to optimally fit the data. The result of using this approach is compared with the result obtained using a rigid-body description of the CYP3A4-in-nanodisc system. Here, the CYP3A4 structure relies on detailed information from crystallographic and molecular-dynamics studies of CYP3A4. Both modelling approaches arrive at very similar solutions in which the α-helical anchor of the CYP3A4 systematically stays close to the edge of the nanodisc and with the large catalytic domain leaning over the outer edge of the nanodisc. The obtained distance between the globular domains of CYP3A4 is consistent with previously published theoretical calculations.
Nanodiscs are monodisperse, self-assembled discoidal particles that consist of a lipid bilayer encircled by membrane scaffold proteins (MSP). Nanodiscs have been used to solubilize membrane proteins for structural and functional studies and deliver therapeutic phospholipids. Herein, we report on tetramethylrhodamine (TMR) tagged nanodiscs that solubilize lipophilic MR contrast agents for generation of multimodal nanoparticles for cellular imaging. We incorporate both multimeric and monomeric Gd(III)-based contrast agents into nanodiscs and show that particles containing the monomeric agent (ND2) label cells with high efficiency and generate significant image contrast at 7 T compared to nanodiscs containing the multimeric agent (ND1) and Prohance, a clinically approved contrast agent.
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