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. 2013 Dec 3;8(12):e81980.
doi: 10.1371/journal.pone.0081980. eCollection 2013.

Identifying Cytochrome p450 Functional Networks and Their Allosteric Regulatory Elements

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Free PMC article

Identifying Cytochrome p450 Functional Networks and Their Allosteric Regulatory Elements

Jin Liu et al. PLoS One. .
Free PMC article

Abstract

Cytochrome P450 (CYP) enzymes play key roles in drug metabolism and adverse drug-drug interactions. Despite tremendous efforts in the past decades, essential questions regarding the function and activity of CYPs remain unanswered. Here, we used a combination of sequence-based co-evolutionary analysis and structure-based anisotropic thermal diffusion (ATD) molecular dynamics simulations to detect allosteric networks of amino acid residues and characterize their biological and molecular functions. We investigated four CYP subfamilies (CYP1A, CYP2D, CYP2C, and CYP3A) that are involved in 90% of all metabolic drug transformations and identified four amino acid interaction networks associated with specific CYP functionalities, i.e., membrane binding, heme binding, catalytic activity, and dimerization. Interestingly, we did not detect any co-evolved substrate-binding network, suggesting that substrate recognition is specific for each subfamily. Analysis of the membrane binding networks revealed that different CYP proteins adopt different membrane-bound orientations, consistent with the differing substrate preference for each isoform. The catalytic networks were associated with conservation of catalytic function among CYP isoforms, whereas the dimerization network was specific to different CYP isoforms. We further applied low-temperature ATD simulations to verify proposed allosteric sites associated with the heme-binding network and their role in regulating metabolic fate. Our approach allowed for a broad characterization of CYP properties, such as membrane interactions, catalytic mechanisms, dimerization, and linking these to groups of residues that can serve as allosteric regulators. The presented combined co-evolutionary analysis and ATD simulation approach is also generally applicable to other biological systems where allostery plays a role.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Networks detected by co-evolutionary analysis of CYP2C sequences.
A: heat map showing the co-evolutionary score for each pair of residues tagged as evolving together above the evolutionary noise. Not all residues in the CYP2C sequences are shown, and the order of the residues was determined via clustering based on the co-evolutionary score. The residue clustering identified four primary groupings that we characterized as a membrane-binding network, a catalytic network, a dimerization network, and a heme-binding network. Although the networks are separable by their primary function, cross-talk can occur between these networks as is evident from the off-diagonal elements. Each pairwise entry was color coded from high (red) to low (blue) co-evolutionary scores. Each residue network was further mapped to the atomic structure of CYP2C9 (PDB code 1R9O) in B-E and shown in the following different orientations: the membrane-binding network (B), the catalytic network (C), the dimerization network (D), and the heme-binding network (E). The heme molecule is shown in a stick representation, and the residues belonging to the functional networks are shown as spheres.
Figure 2
Figure 2. Membrane-binding networks for different CYP isoforms.
Residues identified as belonging to membrane-binding networks were mapped to the molecular structures of CYP1A (PDB code 2HI4; A), CYP2C (PDB code 1R9O; B), CYP3A (PDB code 1W0E; C), and CYP2D (PDB code 2F9Q; D). The location of the membrane-bound portion of the enzyme partly determines which substrates can access each enzyme. The heme molecule is shown in a stick representation, and the residues belonging to the functional networks are shown as spheres. The position of the membrane line was qualitatively positioned based on the location of the residues in the membrane-binding networks.
Figure 3
Figure 3. Molecular components of the catalytic network of the CYP3A4-adrenodoxin (Adx) complex.
Adx is a small iron-sulfur protein that transfers electrons from NADPH to terminal CYP as part of the overall metabolic processing of substrates. We used the structural complex of this enzyme with eukaryotic CYP (CYP11A1) to model a CYP3A4-Adx complex. Selected molecular components of the modeled electron transfer pathway from the CYP3A4 heme iron to the Adx are shown, where the CYP3A4 unit is shown in gray, the Adx unit is shown in yellow, and the residues in the CYP3A4 catalytic networks are shown in green. The heme group is shown in pink, and the CYP3A4 C411 residue is shown in red. All residues involved in the predicted electron-transfer pathway are shown as sticks.
Figure 4
Figure 4. Functional networks of CYP2C9.
Dimerization and membrane-binding networks were mapped on the crystal structure model of a CYP2C9 dimer (PDB code 1R9O). The dimerization network is shown in pink, the membrane-binding network is shown in orange, and the heme group is shown in magenta on the right monomer. The F-G loop is shown in yellow. The position of the membrane line was qualitatively positioned based on the location of the residues in the membrane-binding networks.
Figure 5
Figure 5. Co-evolved residues in the heme-binding networks.
Residues identified as belonging to heme-binding networks were mapped to the molecular structures of CYP1A (PDB code 2HI4; A), CYP2C (PDB code 1R9O; B), CYP3A (PDB code 1W0E; C), and CYP2D (PDB code 2F9Q; D). The heme molecule is shown in stick representation, and residues belonging to the functional networks are shown as yellow spheres. Note that this network consists of both co-evolved residues that are in direct proximity to the heme as well as residues that are located distal to the heme.
Figure 6
Figure 6. CYP3A4 interactions and metabolic fate of Nile red (NR).
We docked the NR substrate to the CYP3A4 enzyme to identify how the substrate interacts with the heme group in the active site. The two top-scoring poses of NR are show in A, where the NR molecule is shown as sticks in orange (pose 1), and in B, where it is depicted in yellow (pose 2). Heme is shown as magenta sticks, residues F304 and A305 are shown as red spheres, and the rest of the enzyme is shown in green. C: possible reaction steps catalyzed by CYP3A4 in the conversion of NR to product 1 (P1) and product 2 (P2). The product ratio of P1 to P2 will depend on how the NR molecule and intermediate metabolites are positioned in the binding pocket.
Figure 7
Figure 7. Residue-based root mean square fluctuations (ΔRMSFs).
ΔRMSFs were calculated from low-temperature anisotropic thermal diffusion (ATD) simulations to identify residues that exhibit long-range correlated motions to a locally heated heme group in the CYP3A4 structure. A: WT CYP3A4 residues that interact with NR are labeled as numbers or number ranges and partially overlapped with the ΔRMSF peaks. Key residues 301-306, which provide the means to change the NR reaction products, are shown in red. B and C: ΔRMSFs for the F419A mutant compared with the WT (B) and for the W407A mutant compared with the WT (C). The sequence range that encompasses 301-306 is indicated by blue ovals. All residue numbers refer to the CYP3A4 sequence (PDB code 1W0E).

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Grant support

Funding of this research was provided by the U.S. Department of Defense Threat Reduction Agency Grant TMTI0004_09_BH_T. The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the US Army or the US Department of Defense. This paper has been approved for public release with unlimited distribution. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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