Influence of Transmembrane Helix Mutations on Cytochrome P450-Membrane Interactions and Function

Abstract

Human cytochrome P450 (CYP) enzymes play an important role in the metabolism of drugs, steroids, fatty acids, and xenobiotics. Microsomal CYPs are anchored in the endoplasmic reticulum membrane by an N-terminal transmembrane (TM) helix that is connected to the globular catalytic domain by a flexible linker sequence. However, the structural and functional importance of the TM-helix is unclear because it has been shown that CYPs can still associate with the membrane and have enzymatic activity in reconstituted systems after truncation or modification of the N-terminal sequence. Here, we investigated the effect of mutations in the N-terminal TM-helix residues of two human steroidogenic enzymes, CYP 17A1 and CYP 19A1, that are major drug targets for cancer therapy. These mutations were originally introduced to increase the expression of the proteins in Escherichia coli. To investigate the effect of the mutations on protein-membrane interactions and function, we carried out coarse-grained and all-atom molecular dynamics simulations of the CYPs in a phospholipid bilayer. We confirmed the orientations of the globular domain in the membrane observed in the simulations by linear dichroism measurements in a Nanodisc. Whereas the behavior of CYP 19A1 was rather insensitive to truncation of the TM-helix, mutations in the TM-helix of CYP 17A1, especially W2A and E3L, led to a gradual drifting of the TM-helix out of the hydrophobic core of the membrane. This instability of the TM-helix could affect interactions with the allosteric redox partner, cytochrome b5, required for CYP 17A1's lyase activity. Furthermore, the simulations showed that the mutant TM-helix influenced the membrane interactions of the CYP 17A1 globular domain. In some simulations, the mutated TM-helix obstructed the substrate access tunnel from the membrane to the CYP active site, indicating a possible effect on enzyme function.

Differing Membrane Interactions of Two Highly Similar Drug-Metabolizing Cytochrome P450 Isoforms: CYP 2C9 and CYP 2C19

“…CYP 2C9 mainly adopted a class A orientation, which has lower α, β and heme-tilt angles, and CYP 2C19 adopted a class B orientation. The class B orientation is similar to the orientations observed in simulations with the same bilayer and force field for CYP 3A4 and N-terminal mutants of CYP 17A1 and CYP 19A1, with measured heme-tilt angles of about 60° [27]. CYP 2C9 thus appears to be unusual in this set of CYPs (simulated under the same conditions) in adopting an orientation with a lower heme-tilt angle.…”

“…These differences can be attributed in large part to differences in both the protein and the lipid force field used. Our studies have shown that the force field used in the current work produces results in better agreement with experimental data for simulations of CYP-membrane systems, including excellent agreement with linear dichroism measurements of the heme-tilt angle of CYPs in Nanodiscs [25,26,27].…”

“…The orientation of the CYP globular domain in a membrane may be affected by simulation parameters such as the force field used and the procedure used to model and sample the conformational space of the simulated systems. We have shown previously that the force field and procedure employed here can give good agreement with experiments [25,27]. However, other factors relevant in vivo may affect the orientation of the CYP globular domain, such as homodimerization [37], heterodimerization, or the binding of redox partner proteins.…”

Use of Computational Biochemistry for Elucidating Molecular Mechanisms of Nitric Oxide Synthase

“…Numerous experimental data are available on the structure of the NOS heme domain in the closed state, but several aspects remain to be elucidated. For comparison with a homolog, extensive in silico studies (e.g., microsecond all-atom and coarse-grain MD, docking) have been reported on P450 enzymes [ [73] , [74] , [75] , [76] , [77] , [78] ], including the investigation of the structure of the active site, substrate tunneling and binding modes, mutational effects, and interaction with membranes. These show the usefulness of molecular modeling in a context similar to NOSox.…”

Structural and Functional Analysis of Tomato Sterol C22 Desaturase

“…The globular domain of tomato C22DES interacts with the ER in the absence of TMH It has been reported that the globular domain of several CYP proteins interacts with the ER membrane in the absence of the transmembrane domain (32)(33)(34)(35). To determine if this is the case in tomato C22DES, a N-terminal truncated form of the enzyme lacking the TMH region (residues 2 to 27) was fused to the Nterminal end of RFP and the resulting protein (C22DESΔ2-27-RFP) transiently expressed along with TMH-GFP in N. benthamiana leaves.…”