Membrane-Anchored Cytochrome P450 1A2âCytochrome b5

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Membrane-anchored cytochrome P450 1A2–cytochrome b5 complex features an X-shaped contact between antiparallel transmembrane helices Petr Jerabek, Jan Florian, and Václav Martínek Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00349 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on February 29, 2016

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Chemical Research in Toxicology

Membrane-anchored cytochrome P450 1A2–cytochrome b5 complex features an X-shaped contact between antiparallel transmembrane helices

Petr Jeřábek†, Jan Florián‡, Václav Martínek†,§* †

Department of Biochemistry, Charles University in Prague, Faculty of Science, Albertov 2030, 128 43 Prague 2, Czech Republic

‡

Department of Chemistry and Biochemistry, Loyola University Chicago, 1032 W. Sheridan Rd., Chicago, IL 60660, USA

§

Department of Teaching and Didactics of Chemistry, Charles University in Prague, Faculty of Science, Albertov 3, 128 43 Prague 2, Czech Republic

E-mail: [email protected]

Running header Prediction of the membrane-anchored cytochrome P450 1A2–cytochrome b5 complex

KEYWORDS cytochrome b5, cyt b5, cytochrome P450 1A2, CYP1A2, protein-membrane interactions, membrane proteins, protein–protein interactions; transient interactions, protein-membrane self-assembly, coarse-grained molecular dynamics, molecular dynamics, MARTINI force field, molecular modeling

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ABSTRACT Eukaryotic cytochromes P450 (P450) are membrane bound enzymes oxidizing a broad spectrum of hydrophobic substrates including xenobiotics. Protein–protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates catalytic activities of several P450s. To lay structural foundation for the investigation of these effects we constructed a model of the membrane-bound full-length human P450 1A2–cyt b5 complex. The model was assembled from several parts using multi-scale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2– cyt b5 complexes, these simulations yielded three stable binding modes of (sAI, sAII, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mAI and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mAII mode was found to be unstable in the membrane environment. The mutual position of soluble domains in the binding mode mAI was analogous to the sAI complex. Featuring the largest contact area, the smallest structural flexibility, the shortest electron transfer distance and the largest number of inter-protein salt bridges, the mAI mode is the best candidate for the catalytically relevant full-length complex.



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INTRODUCTION Eukaryotic cytochromes P450 (P450) are membrane bound enzymes playing a key role in the oxidation of a broad spectrum of hydrophobic substrates, e.g. xenobiotics, drugs, sterols, and fatty acids. P450 are frequently anchored on the cytosolic side of the endoplasmic reticulum.1,2 Catalytic activity of P450 depends on a sequential transfer of two electrons, which are delivered by a membrane-bound NADPH:P450 reductase or NADPH:P450 reductase together with another membrane-bound protein, cytochrome b5 (cyt b5).3 Cyt b5 is a small membrane hemoprotein that can, in addition to being an electron donor, enhance, inhibit, or have no effect on the catalysis by P450. The actual outcome of the cytochrome P450–cyt b5 interaction depends on the particular P450 enzyme, its substrate, or the experimental conditions.4,5 Interestingly, cyt b5 can also significantly alter the ratio of products formed during oxidation of benzo[a]pyrene and anticancer drug ellipticine catalyzed by P450 1A1, 1A2, and 3A4.6–9 The important role of cyt b5 in the P450 mediated drug metabolism was also confirmed during in vivo experiments with humanized mice carrying a hepatic cyt b5 deletion.10 Therefore, knowledge of the complex structure is important for elucidating the mechanism of the cyt b5-mediated P450 modulation, which may involve allosteric effects.11–13 Inter-protein contacts in complexes of cyt b5 with a P450 in the membrane environment were studied using indirect experimental techniques, such as site-directed mutagenesis, NMR spectroscopy and cross-linking experiments,14,15,15–21 leading to the consensus that the convex and acidic surface of cyt b5 binds to the basic concave surface of a P450 protein.22 In the absence of experimental three dimensional structures23 we recently used computational methods to propose structure of the complex between soluble domains of human P450 1A2 and cyt b5.24 However, the membrane environment, which was suggested to


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strengthen the cytochrome P450–cyt b5 interaction25 via stabilizing their membrane-associated domains26, is required for activity of eukaryotic cytochromes P450.27,28 Steady improvements in computer performance, new computational algorithms, and biomolecular force fields have vaulted molecular dynamics (MD) simulations into position of an indispensable tool for investigating structure and dynamics of membrane proteins.29–31 Unfortunately, MD simulations of membrane proteins are frequently complicated by absent or insufficient initial structural information on the lipid molecules or detergents surrounding the membrane domain. Consequently, significant errors may arise when the initial guess is based on indirect experimental information32 or implicit membrane predictions.33 The insufficient initial structural insight could be mitigated by “coarse-grained” (CG) self-assembly techniques, which proved to be well suited for finding the “sweet spot” of a membrane protein within its natural environment, even when the initial guess is far from the optimum.34–36 Allatom and/or CG MD simulations were prominently featured in studies of the self-assembly and dynamics of antimicrobial, viral, and synthetic membrane peptides,35,37–39 high-density lipoproteins,40–42

mechanoselective

proteins,43–46

G

protein-coupled

receptors,47–49

rhodopsin,50–52 voltage gated cationic channels,34,53–57 and ligand- or proton-gated ion channels,58–60 and cytochromes P45026,32,61–67 The realism of CG simulations and their relevance for the experimental scientific community can be further elevated by multi-scale modeling that combine all-atom and CG MD approaches,26,50,68,69 including forays into realm of free energies.70 In this paper, we applied multi-scale modeling techniques in a new way and improved the P450 1A2–cyt b5 model by reconstructing both proteins in full-length (containing their membrane anchors) and by explicit consideration of the phospholipid membrane environment. Specifically, we combined the structures of the soluble complexes with the spontaneously



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assembled CG models of their transmembrane (TM) domains, and followed the dynamics of the full-length all-atom complex.


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METHODS CG model of the membrane-bound complex of P450 1A2 and cyt b5 was constructed by combining an in silico structure of the complex of their soluble domains24, with the structure of the membrane bound P450 1A2, and the end point of our CG MD self-assembly of the cyt b5 TM domain. Upon the completion of CG MD simulations and conversion from the CG to all-atom model, three most viable conformers of the membrane-anchored complex were subjected to extended all-atom MD simulations to elucidate their most stable 3-D structure (Figure 1). The protonation states of ionizable moieties were assigned according to their pKa in solution. The protonation states of histidine residues were assigned manually using chemical intuition (Table S1).

General setup of CG and all-atom MD simulations CG MD simulations were performed using the MARTINI 2.1 CG force field71,72 implemented in NAMD v2.9.73 This force field typically joins four heavy atoms and their hydrogens into a single particle (bead), thus reducing number of simulated particles, allowing a large step-size (up to 25 fs), and lowering friction inside the simulated system.. One backbone bead represents all backbone atoms of each amino acid residue; larger residues also include side chain beads. The parametrization of four basic types of CG particles (charged, polar, nonpolar, and apolar) takes into account experimental polar/apolar partition coefficients. Additional particle subtypes are used to simulate H-bonding capabilities. All CG simulations were carried out under periodic boundary conditions in the isothermal–isobaric (NPT) ensemble (310 K, 1 atm, constant number of particles). Temperature and pressure were held constant using Langevin dynamics (friction coefficient 1) and Langevin piston method. Isotropic and semi-isotropic pressure coupling were used in the simulations of the phospholipid bilayer self-assembly and the membrane bound P450 1A2–cyt



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b5 complex, respectively. The integration time step was 20 fs. Lennard–Jones and electrostatic interactions were shifted to zero for interatomic distances in the 9 - 12 Å, and from 0 - 12 Å range, respectively.74 Based on the earlier cytochrome P450 implementation26 of the elastic network model75, we used a 10.75 kcal/(mol Å2) harmonic force constant to constrain distances between backbone beads that were in the corresponding crystal structures closer than 7 Å. These constraints allowed retaining the mutual orientation and the secondary/ternary structures of the soluble P450 1A2 and cyt b5 domains during CG MD simulations. All-atom MD simulations were carried out in an explicit solvent under periodic boundary conditions in the NPT ensemble (310 K, 1 atm), using CHARMM2776 and its update for lipids (CHARMM3677). Temperature and pressure were held constant using Langevin dynamics (friction coefficient 1) and Langevin piston method implemented in program NAMD v2.9.73 The simulation time step was 2 fs. Lennard–Jones interactions were shifted to zero for interatomic distances between 9 and 12 Å, and cut-off for electrostatic interactions was 12 Å.74 For long-range electrostatic interactions, we used the particle mesh Ewald method78 implemented in NAMD. Bonds involving hydrogen atoms and TIP3P water were kept rigid using SHAKE algorithm.79

Self-assembly of the cyt b5 TM domain/DLPC/water system (Figure 1, step 1) The simulated system consisted of a 103 Å cubic box containing the human cyt b5 TM domain (residues 94-134) that was immersed in 5149 water molecules and 350 randomly placed molecules of DLPC (dilauroylphosphatidylcholine). DLPC and water molecules were randomized using software PACKMOL.80 Initial all-atom coordinates for the cyt b5 TM domain were generated using PyMol. The initial secondary structures were assigned as coil (N94-N112 and M131-D134) or helix (W113-Y130) according to predictions made by PSIPRED and TMHMM servers.81,82 Using VMD83, 17 K+ and 14 Cl- ions were additionally


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uniformly distributed in the cubic box to mimic 0.1M KCl solution and neutralize the simulated system. The resulting all-atom model of the protein/lipid/water/ion mixture was converted to the MARTINI CG model.71,72 NAMD v2.973 was used to minimize the system energy (1000 steps) and to run 10 independent 100 ns CG MD simulations. The population analysis of the TM cyt b5 helix orientation was performed for the last 68 ns of one randomly selected trajectory.

Assembly and CG MD simulations of a full-length membrane bound complex of P450 1A2–cyt b5 (Figure 1, steps 2 and 3) For the construction of the CG model of the membrane-anchored complex of P450 1A2 and cyt b5, we needed to overlay and join the previously proposed complex of their soluble domains24 with the recent CG model of membrane bound P450 1A284, and link the resulting structure with the cyt b5 TM domain from our step 1. First, we converted the soluble P450 1A2–cyt b5 complex from the all-atom to the CG representation. This structure was then aligned to the CG model of the full-length P450 1A2 in a membrane via backbone beads of residues 34 to 513 of P450 1A2 (Figure 1, step 2). While preserving cyt b5 TM domain’s tilt and surrounding DLPC molecules, we manually shifted this domain inside the membrane into an approximate distance of 45 Å from the TM domain of P450 1A2, which was the maximal distance permitted by the length of the cyt b5 linker. The large initial separation of TM domains eliminated any specific contact predispositions, thus allowing us to examine the spontaneous formation of the complex. The simulated system consisted of a periodic box (147 x 107 x 177 Å) containing both membrane proteins that were immersed in a phospholipid bilayer containing 475 DLPC molecules and 13466 water molecules. 42 K+ and 44 Cl- ions were additionally uniformly distributed in the cubic box to mimic 0.1M KCl solution and neutralize the simulated system.



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Both hemes were removed from the complex, as no CG parameters were available for them. Due to the elastic network constraints, the absence of the heme did not influence the tertiary structure of P450 1A2. For cyt b5, an additional constraint (equil. distance 4.83 Å, force constant 50 kcal/(mol Å),) between side chain beads (SI1 of H44 and SI1 of H68) of heme ligands was applied to compensate the heme absence. A 7 µs long production phase (Figure 1, step 3) was preceded by a multistep equilibration (Table S2).

Conversion of CG models to all-atom models (Figure 1, step 4) The conversion of the CG model to its atomic coordinates was performed for the most frequently occurring conformation of the membrane bound P450 1A2–cyt b5 complex. To identify this conformation a set of ten geometric parameters, which relate rigid-body positions of protein domains and the membrane, were devised and measured for every 50,000th CG MD step. These parameters included distances from the center of the phospholipid bilayer to the center of mass of the soluble P450 1A2 domain (d1), TM cyt b5 domain (d2), and TM P450 1A2 domain (d3). Furthermore, four auxiliary vectors (v1, v2, v3 and v4) of important helixes were used to define angles α, β, γ, and ζ between these vectors and an axis perpendicular to the phospholipid bilayer (z-axis) (Figure 2). We also took into consideration the mutual orientation of the P450 1A2 soluble and TM domains (angles δ and ε) and the mutual orientation of the TM domains of P450 1A2 and cyt b5 (angle η). Parameters d1, d2, d3, α, β, γ, δ , ε, ζ and η were set to their most populated values (Figure S1). The resulting representative CG structure of the P450 1A2–cyt b5 complex was converted to its atomic representation using algorithm of Shih et al.85 and CGTools integrated in VMD v1.9.183. Further details on the conversion of CG models to all-atom models are included in Supporting Information.


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The coordinates of the P450 1A2 TM domain (residues 1 to 33), the cyt b5 TM domain (residues 93 to 134), and the phospholipid bilayer were obtained by the CG to all-atom conversion. In order to prevent distortions of the tertiary structure of P450 1A2 globular domain, its atomic coordinates (residues 34-513) were taken from X-ray diffraction data (PDB ID 2HI4). The orientation of cyt b5 with respect to P450 1A2 was repositioned to represent modes sAI, sAII, and sB from our earlier all-atom simulations of the soluble P450 1A2–cyt b5 complex.24 The representative structures of the sAI, sAII, and sB modes of the soluble complex were aligned to the all-atom model of the membrane bound complex via backbone atoms of P450 1A2 (residues 34-513). However, some charged side chains of P450 1A2 residues, which were in close contact with cyt b5, adopted significantly different orientation in their crystallographic positions than in complexes represented by binding modes sAI, sAII, and sB. This difference in side-chain alignment might result in sterical collisions with cyt b5 and distortion of salt bridges formed during optimization of individual binding modes. Therefore, a set of constrains was applied on those sidechains during the equilibrations to restore salt bridges of the membrane bound models (Table S3).

All-atom MD simulations All-atom MD simulations were carried out to sample conformational space of the soluble P450 1A2–cyt b5 complex on the microsecond time-scale. The simulated system consisted of a 117 Å cubic box containing the protein complex surrounded by 29390 water molecules. 26 K+ and 29 Cl- ions were uniformly distributed in the box to mimic 0.1M KCl solution and to neutralize the simulated system. A docked structure of the soluble complex24 was used to initiate these simulations, which were subsequently branched into six separate trajectories (t1t6) having a combined length of 2 µs (t1 475 ns, t2 388 ns, t3 270 ns, t4 270 ns, t5 270 ns, and t6 270 ns). The resulting conformations were clustered based on the root-mean-square



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deviation (rmsd) of their cyt b5 backbone atoms from the initial docked structure24 after each conformation of the complex was aligned to the initial structure of P450 1A2. The clustering was performed using RMSD Trajectory Tool in VMD with 1 Å cut-off to bin individual snapshots. The structures representing binding modes sAI, sAII and sB were determined from trajectory fragments t6 (20–270 ns), t3 (10–270 ns), and t1 (230–475 ns), respectively, as follows: Each snapshot on a trajectory fragment was first aligned to the initial structure from protein-protein docking.24 Subsequently, a trajectory-averaged geometry was calculated for each binding mode from these aligned geometries. Finally, a snapshot geometry that had the smallest rmsd from this trajectory-averaged geometry became the representative structure for the corresponding binding mode. All-atom models of membrane bound P450 1A2–cyt b5 complexes in modes sAI, sAII, and sB were solvated using software VMD v1.9.183. Water molecules present in the crystallographic coordinate files were preserved except for waters located at the interface of both proteins. The simulated system consisted of a periodic box (126 x 113 x 132 Å) containing the protein complex immersed in a phospholipid bilayer containing 475 DLPC molecules and 34397 water molecules. 97 K+ and 99 Cl- ions were uniformly distributed in the cubic box to mimic 0.1M KCl solution and neutralize the simulated system. Each protein complex was equilibrated in several steps (Table S4). Subsequently, each binding mode was subjected to ten independent 46 ns MD simulations. Representative structures were selected from these simulations using an approach described in the preceding paragraph. Distribution of electron donor−acceptor distances, buried surface areas, and percentage occurrences of salt bridges between soluble domains of P450 1A2 and cyt b5 were calculated from the same set of trajectories that were used to determine representative structures (vide supra). Mutual orientation of TM domains was described using angular distributions of parameters γ, ζ and η (Figures 2 and 8). These parameters were evaluated


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from all 20 trajectories (combining modes mAI and mB), while excluding first 10 ns from each trajectory.



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RESULTS AND DISCUSSION Orientation of the cyt b5 TM domain in phospholipid bilayer Multiple CG MD trajectories were generated to simulate the TM domain of the human cyt b5 that was initially immersed in a randomized mixture of water and DLPC molecules. Approximately half of these ten 100 ns simulations resulted in the formation of a phospholipid bilayer that was spanned by the TM helix (Figure 1, step 1). The spontaneous self-assembly is illustrated for one of these productive trajectories in Supporting Information (Movie S1). The final segment of the same trajectory, which sampled dynamics of cyt b5 TM helix in the already assembled membrane, was used to examine the TM cyt b5 helix orientation with respect to the phospholipid bilayer. The analysis revealed that the TM helix preferred tilts of 30° and 45° (Figure 3). After the correction for the membrane thickness, these values compare favorably with NMR experiments for the rabbit cyt b5 TM helix in DMPC/DHPC bicelles.86 That is, the membrane associated C-terminal domain of rabbit cyt b5 differs from its human ortholog only in five conservative amino acid residues (Figure S3 in SI), but the dimyristoylphosphatidylcholine (DMPC) phospholipid bilayer used for the experiment is significantly thicker than the DLPC bilayer. Therefore, we used simple trigonometric manipulations to adjust the reported experimental tilt value for thickness difference between the DMPC and DLPC membranes (Figure S4 in SI). The resulting adjusted experimental value of the TM helix tilt (34 ± 3°) is close to one of the preferred angular orientations from our CG MD simulations. Therefore, we used the TM helix of cyt b5 tilted by a 30° to initiate the full-length protein CG simulation (Figure 1, step 2).

Antiparallel TM helices spontaneously associate during CG simulations Observations of Yamamoto et al. suggested that the TM helix of cyt b5 in complex with rabbit P450 2B4 was dynamically restrained and could directly interact with the P450 TM domain.86


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Because there is currently no experimental structural information about mutual orientation of TM helices of P450 and cyt b5, we setup our simulations to allow spontaneous association of these helices. The two TM helices, which were covalently attached to their soluble domains via peptide linkers, were initially positioned ~45 Å apart in an antiparallel orientation. The unstructured cyt b5 linker spontaneously attached to the surface of P450 1A2 within the first ~100 ns of the unconstrained CG simulation. This linker attachment was followed by the gradual spontaneous association of P450 1A2 and cyt b5 TM helices during the subsequent ~100 ns simulation (Figure 1, step 3 and Figure 4). The TM helices remained in contact for additional ~6,800 ns, yielding a dynamic X-shaped complex that was not stabilized by any specific amino acid interactions. In order to assess potential influence of the cyt b5 peptide linker on the TM helices self-association we performed an additional simulation, in which the linker between cyt b5 soluble and TM domain was interrupted and its major portion (residues 93 to 108) was removed. This control simulation also yielded spontaneous association of the P450 1A2 and cyt b5 TM helices (data not shown), indicating that the driving force for the TM-TM helix self-association is generated by the membrane domains themselves and/or is mediated by a specific structure of the local membrane environment. Due to a large complexity of the membrane bound P450 1A2–cyt b5 system, we employed a set of seven geometric parameters to facilitate selecting a representative CG structure for the conversion to the all-atom representation. Three of these parameters were originally used by Cojocaru at al. to characterize the P450 2C9 orientation in the phospholipid membrane.26 Here, we introduced four additional parameters describing the mutual orientation of the P450 1A2 and cyt b5 TM helices and the phospholipid bilayer (Figure 2). The calculated distribution of these parameters was used to identify a structure embodying the most frequently occurring coordinates (Figure S1).



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Number of distinct binding modes of soluble domains is reduced in the full-length complex To generate robust reference for the analysis of the membrane-bound complex, we carried out six all-atom MD simulations, t1 – t6, that sampled the conformational space of the soluble P450 1A2–cyt b5 complex in aqueous solution on the microsecond time-scale. The calculated rmsd distribution showed that the sampled soluble complex conformations fall into three separate groups, with their rmsd values near 6, 13, and 20 Å (black line in Figure 5). These groups are, respectively, denoted as modes sAI, sAII, and sB. The conformation of soluble domains in the sAI mode resembles the structure obtained previously by protein-protein docking.24 While being observed in the early part of all simulations, the sAI mode dominated trajectories t4 and t6 (Figure S2). The complex in two other simulations (t3 and t5) switched after 10 ns to the mode sAII, which is structurally more distant from the initial structure (rmsd = 12 Å, Table 1). The simulation t5 showed that the sAII mode is sufficiently stable (for ~250 ns) to be recognized as a stable complex of soluble domains. In addition, the oscillations between modes sAI and sAII observed during the t3 trajectory indicated that the barrier between these modes is relatively small and the transition between these two conformations is reversible (Figure S2). Mode sB is the most distant from the starting structure (rmsd = 21 Å, Table 1). This mode was visited by cyt b5 in simulations t1 and t2 after about 240 ns. In order to examine the stability of mode sB, trajectories t1 and t2 were extended by 200 and 100 ns, respectively. In both cases, the complex remained stable for entire duration of the added simulation time. Although the total simulation time of 1.4 µs (500, 520, and 383 ns for modes sAI, sAII, and sB, respectively) was too short for evaluating relative free energies of individual binding modes, it provided sufficient sampling for their structural and dynamic characterization using our heme-centric internal coordinates (Figure 2 in Jeřábek at al, Biochemistry, 2014)24 and 16 Environment ACS Paragon Plus

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their standard deviations (Table 1). In particular, we noticed that the sAI mode fluctuated less than modes sAII or sB. Structures representing all three binding modes were further used for examining their viability in the presence of TM domains and for constructing initial models of the membrane bound P450 1A2–cyt b5 complex. Our models of the soluble P450 1A2–cyt b5 complex in modes sAI, sAII, and, sB were extended to more physiologically relevant full-length membrane-anchored protein models, mAI, mAII, and, mB. Despite the large size of these all-atom MD systems (~150,000 atoms), our total simulation time reached 460 ns. In mode mAII, the tension in the 20-aminoacid linker that connects the cyt b5 soluble domain with its TM helix turned out to be incongruent with the stability of the complex. This tension was caused by the large distance between the C- and N-termini of these domains. Consequently the mAII mode was transformed into mode mAI already during the 4 ns long equilibration phase (Figure S5) resulting in the absence of the peak at ~13 Å for the membrane-anchored complex (Figure 5). Since all MD trajectories that were initiated from mode mAII turned out to sample only the conformational space of mode mAI we did not initiate any new trajectories from the mode mAI. Although all trajectories that sampled mAI or mB complexes showed considerable atomic fluctuations, there is zero population size for rmsd from 11 to 17 (Figure 5); therefore these fluctuations did not result in switching between these two binding modes. Previously, we supposed that the membrane environment would have no dramatic effect on residues involved in the P450 1A2–cyt b5 protein–protein interaction interface;24 this assumption is now supported by the observed large distance between soluble domain of cyt b5 and the phospholipid membrane (Figure 6). Specifically, the closest amino acid residues of this domain reside in modes mAI and mB about 23 and 18 Å above the membrane, respectively. The comparison of soluble domains of P450 1A2 and cyt b5 in soluble (sAI, sB) and membrane-anchored (mAI, mB) complexes showed only minor differences. In particular, 17 Environment ACS Paragon Plus


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the mutual position of soluble domains in the membrane bound mode mAI is very similar to the structure of the soluble P450 1A2–cyt b5 complex that was predicted using extensive protein–protein docking combined with classical and steered MD24, and further refined here using multiple MD simulations (Table 1, Figure 5). On the other hand, sB and mB modes show shifted heme-centric internal coordinates and rmsd (Table 1, Figure 5). The 1 Å rmsd structural differences between sB and mB modes could be due to higher flexibility of the P450 contact surface in the B mode than in AI mode. The AI contact interface is formed mainly by relatively rigid B and L helices that are characterized by an average root mean square fluctuation (rmsf) of 0.9 ± 0.3 Å. In contrast, the B mode contact is formed by more dynamic helices C, H, and loop HI (rmsf = 2.5 ± 0.6 Å) (Figure S6). Electrostatic complementarity plays significant role in the interaction between P450 1A2 and cyt b5.24 Here we mapped the occurrence of salt bridges at the interface of the P450 1A2 and cyt b5 soluble domain, explicitly listing those that lasted for over 20% of the simulation time of each mode (Figure 7). Ten salt bridges intermittently formed in the sAI mode complex comprising of only soluble domains. Eight of these salt bridges were found also in the full-length mAI mode complex (Figure 7 and Figure S8). Most of these bridges were described in our previous study.24 The R34-E48 pair was formed only in the sAI mode where R34 is actually the N-terminal residue. In the full length P450 1A2 model, R34 is positioned in the linker connecting the catalytic and TM domains which restrain this residue to the membrane bound region that is far from the cyt b5 soluble domain. In total, ten salt bridges were detected for the B modes; however only four of these salt bridges were shared by the sB and mB modes (Figure 7). The lower similarity between B modes is probably caused by their higher instability, which result in a frequent realignment of interprotein salt bridges in the contact interface. Thus, compared to the mAI mode, mode mB has a lower number of stable salt bridges. In addition, the absence of common salt bridges in


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the mAI and mB binding modes indicates that cyt b5 forms contacts with different areas of P450 1A2 surface in these modes. The elevated percentage of charged residues that form the interface between P450 1A2 and cyt b5 in the membrane-bound modes mAI (nonpolar 40.0%, polar 3.3%, charged 56.7%) and mB (nonpolar 44.8%, polar 17.2%, charged 37.9%) is in accordance with the study of Ansari and Helms87, who analyzed a set of predominantly transient protein-protein complexes and found similar ratios of interfacial residues (nonpolar 30.4%, polar 32.8%, charged 36.8%). A highly charged protein–protein interface would be expected in a transient complex similar to P450 1A2–cyt b5, which needs to quickly bind/unbind. A more frequent occurrence of hydrophobic residues would only contribute to a larger affinity that is not desirable in this type of interaction.

Interaction of P450 1A2 and cyt b5 with membrane environment is similar in modes mAI and mB In the mAI and mB modes, a major portion of the P450 1A2 TM helix (residues L14-L28) and the W48, P49, L50, L51, F239, I241, Y244, and L245 residues of its globular domain are buried within the hydrophobic part of the phospholipid membrane. In both modes, only the cyt b5 TM helix residues V114-Y127 interact with the hydrophobic membrane segment. All these findings indicate that the mAI and mB modes substantially deviate in the mutual orientation of their soluble domains (Figure 6). The transition between modes mAI and mB in the membrane environment was not observed on the applied simulation time scale, but apparently this transition would only require a ~10 Å translation that is combined with slight rotation of the cyt b5 soluble domain. The recently published data, which were based on photo-initiated cross-linking mapping of the protein–protein interface between membraneembedded portions of rabbit cytochromes b5 and P450 2B488, are consistent with our model of interacting TM helices. Using diazirine analog of methionin (in position 126 of cyt b5) Ječmen



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et al88 found an interaction of this analog with the N-terminal peptide of P450 2B4. The equivalent moieties are in close proximity (~12 Å) in our models mAI and mB.

Contact of antiparallel TM helices is preserved in all-atom simulations The X-shaped contact of TM domains of P450 1A2 and cyt b5, which was formed during their spontaneous association in the CG simulation, was retained during the all-atom simulations. The dynamics of the interacting TM domains was similar for the mAI and mB binding modes. Although TM domains did not dissociate in any of the 20 independent all-atom MD trajectories of the full-length complexes they showed a wide variation of the angle between the two TM helices (Figure 8, angle η). Angles between the phospholipid bilayer and the TM P450 1A2 or TM cyt b5 domain were also found to fluctuate by ±15° from their average values of 42 and 122 Å, respectively (Figure 8, angles γ and ζ). The TM helix-helix interface itself was very fluid, always involving approximately half of residues from segments S10-F25 and I115-M131 of P450 1A2 and cyt b5, respectively. Consequently, no specific residue-pair remained stable during the simulation.

mAI mode has more interfacial salt bridges, larger surface area, and is better configured to support fast electron transfer than mode mB 14 Å electron donor−acceptor distance (rda) is sufficient to facilitate efficient electron transfer between two redox centers embedded in a protein medium.89 MD simulations of the AI binding mode of the soluble and membrane bound complexes yielded identical average rda of 11.8 ± 0.7 Å. In contrast, an rda of 14.2 ± 0.7 and 15.7 ± 0.7 Å, which was obtained for the sB and mB modes, respectively, is less optimal for facile electron transfer (Figure 9) yielding an electron transfer rate that is approximately 100-times slower than for AI modes.


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Not only the number of salt bridges and hydrogen bond contacts, but also the buried surface area is important for estimating protein–protein complex stability. The contact area of the sAI mode is larger than that of the mAI, sB or mB modes (1760 vs 1460, 1200 or 1120 Å2, respectively) (Figure 9). In all cases, the interface between the soluble domains of P450 1A2 and cyt b5 might be classified as a single-patch “standard size” interface according to Lo Conte et al,90 who calculated the “standard size” having a value 1600 ± 400 Å2, for proteaseinhibitor and antibody-antigen complexes. In addition, the areas of the contact interfaces between P450 1A2 and cyt b5 of modes AI and B fall into the 956 – 1852 Å2 range, which was measured for a set of crystal structures of weak transient homodimer complexes.91 The size of the interface is also similar to other short-lived electron-transfer complexes.92

Surface residues that are important for the interaction between selected mammalian cytochromes P450 and cyt b5 are structurally conserved To date, several structural predictions have been attempted for complexes between cyt b5 and P450 2B4, 2E1, 3A4 or 17A1.16,17,20,93–95 These predictions typically used manual or automated protein–protein docking of soluble domains, which were aided by a structural information on several interacting residues obtained from the site-directed mutagenesis, NMR spectroscopy or cross-linking experiments. The majority of resulting models positioned the acidic convex surface of cyt b5 near the basic concave surface of the P450 protein. Besides, all these complexes substantially differed in residue-pairs participating on the inter-protein interaction interface. The pairing differences stemmed mainly from dissimilar cyt b5 rotational orientations with respect to individual P450 proteins. Such large interface variability indicates that experimental information used in these studies might not be sufficient to accurately guide the docking process and/or that the conformational changes needed to reorganize both proteins during complex formation exceed capabilities of the docking algorithms. Consistent



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with this possibility, our final extensively MD optimized model AI shows an rmsd of 6 Å when compared to the initial protein-protein docking structure (Table 1). The discrepancies between published models of P450–cyt b5 complexes could be also attributed to the absence of the membrane environment. This environment might sterically exclude some of the binding modes - a scenario reported here for P450 1A2. Finally, although the catalytic activities of all these P450s are significantly modulated by cyt b5, a possibility that their complexes with cyt b5 might in some cases be significantly structurally different can not be excluded. To address this concern, new experimental structural information on these complexes will be needed. For the mAI model, our MD trajectory analysis identified six positively charged P450 1A2 residues that form salt bridges with cyt b5. Four of these contact residues have structurally equivalent counterparts in P450 2B4, 3A4 and 17A1 that were observed to be important for interaction of these P450s with cyt b5 17,93,95 (Table 2).


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CONCLUSIONS Membrane environment represents an important factor influencing eukaryotic P450 functions. Therefore, we used computer modeling to generate structures of the membrane-anchored P450 1A2–cyt b5 complex. The lack of crystallographic structural information on smaller segments of the whole proteins and the marginal knowledge of their interactions with phospholipids were overcome using multiple spontaneous self-assembly simulations. In the membrane environment, the AI and B binding modes were retained in an essentially unchanged form, whereas the AII mode was excluded as topologically impossible. The mutual position of domains in the mAI binding mode was analogous to the most favorable structure of P450 1A2 and cyt b5 complex identified previously for soluble domains. Based on indirect structure-function descriptors such as the size of the contact area, number of inter-protein salt bridges, structural stability and electron transfer distance we suggest the mAI mode (Figure 6, Table 1) to represent the productive conformation of the full-length P450 1A2–cyt b5 complex. A protein–protein complex formation could be a complex process, in which the rapid formation of an electrostatically-driven initial encounter complex is followed by a slower crawl toward the most stable complex.96 The mB mode, although less stable and more fluctuating than mode mAI, might represent such an encounter complex. The proposed methodology and the predicted structure of the membrane-integrated P450 1A2–cyt b5 complex could aid in structural studies of other cytochromes P450 interacting with cyt b5. It will also serve as an important starting point for the evaluation of the possible allosteric effects induced on P450 1A2 by the presence of cyt b5, including the modulation on the ratios of products observed experimentally for several substrates.6–9



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AUTHOR INFORMATION Corresponding author * Phone: +420 2 21951344. Fax: +420 2 21951283. E-mail: [email protected] Notes The authors declare no competing financial interest. Funding This work was supported by Charles University in Prague (UNCE 204025/2012).

DEDICATION This paper is dedicated to Prof. Marie Stiborová, our outstanding mentor and colleague, on the occasion of her 65th birthday.

ACKNOWLEDGEMENT The authors would like to thank to the Charles University in Prague (grant UNCE 204025/2012). Access to the MetaCentrum and CERIT-SC computing facilities provided under the programs LM2010005 and CZ. 1.05/3.2.00/08.0144 is highly appreciated. VMD was developed by the Theoretical and Computational Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois at UrbanaChampaign.


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ASSOCIATED CONTENT Supporting Information Computational details including additional information on CG and all-atom simulation protocols, conversion of CG models to all-atom models, and the list of constrained salt bridge distances in all-atom simulations of the membrane bound complexes. Visualization and distribution of parameters used for selecting the CG membrane bound P450 1A2–cyt b5 representative structure, time evolution of rmsd of the soluble P450 1A2–cyt b5 complex in all-atom simulations, sequence alignment of soluble domains of human and bovine cyt b5, calculations of tilt angle of cyt b5 TM helix in DLPC membrane, and movie showing typical trajectory of the spontaneous self-assembly of the phospholipid bilayer and cyt b5 TM helix. A coordinate file representing structure of membrane bound P450 1A2–cyt b5 complex in mode mAI. This material is available free of charge via the Internet at http://pubs.acs.org.

ABBREVIATIONS P450, cytochrome P450; cyt b5, cytochrome b5; CG, coarse-grained; MD, molecular dynamics; PDB, Protein Data Bank; rda, donor−acceptor distance; rmsd, root-mean-square deviation; rmsf, root-mean-square fluctuation; DLPC, dilauroylphosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; TM, trans-membrane



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TABLES Table 1. Rmsd and average heme-centric internal coordinatesa of the soluble and membrane– anchored P450 1A2–cyt b5 complexes. Binding mode

Rmsd [Å] b

r [Å]

φ [°]

θ [°]

α [°]

β [°]

γ [°]

sAI

6±1

18.3 ± 0.5

20 ± 4

326 ± 15

30 ± 6

100 ± 7

73 ± 13

sAII

12 ± 2

21.8 ± 0.7

24 ± 7

296 ± 16

40 ± 11

76 ± 35

59 ± 17

sB

21 ± 2

20.7 ± 0.7

32 ± 7

25 ± 34

56 ± 8

120 ± 10

109 ± 8

mAI

6±1

18.3 ± 0.8

20 ± 4

326 ± 29

31 ± 6

99 ± 6

92 ± 9

mB

22 ± 2

36 ± 4

40 ± 6

45 ± 7

104 ± 7

140 ± 8

22.2 ± 0.7

a

Coordinate system introduced by Jeřábek at al.24 was inspired by previous work of Bren97–99.

b

Average values and their standard deviations were calculated from corresponding

trajectories. The rmsd reference was the docked structure published previously.24


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Table 2. Structural conservation of the surface residues that are important for interactions between selected mammalian cytochromes P450 and cyt b5. a Selected cyt b5-sensitive cytochromes P450 P450 1A2 b P450 2B4 P450 2E1 P450 3A4 P450 17A1

a

R95

R85

K87

K91

K88 c

R100

D90

D92

K96 d

K83

R127

S131

R126

e

R138

R126

R362

R343

R344

V350

R347 c

K442

R422

deletion

K424

S427

K447

G425

Y426

deletion

deletion

Structurally and sequentially equivalent residues are show in the common row. Residues that

were found to be important for interactions with cyt b5 are printed in bold. b

P450 1A2 residues forming ionic pairs with cyt b5 in mAI mode. Only residues interacting

for more than 20% of the MD simulation time are listed. c

Important for interaction of P450 17A1 with cyt b5.95

d

Important for interaction of P450 3A4 with cyt b5.93

e

Important for interaction of P450 2B4 with cyt b5.17



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FIGURE LEGENDS Figure 1. Schematic strategy for the assembly of the full-length membrane-bound P450 1A2– cyt b5 complex. The P450 1A2 and cyt b5 proteins in CG model are shown in black and blue, respectively. The P450 1A2 and cyt b5 proteins simulated using all-atom MD are shown in magenta and lime, respectively. For a detailed description of individual steps, see Methods. Figure 2. Definition of vectors (v1-v4) and centers of mass (M1-M3) describing the orientation of the P450 1A2–cyt b5 complex in the phospholipid bilayer. Vector v1 (blue cone) connects one helical turn in helix C and one in helix F, i.e. the center of the Cα atoms of residues 127131 and 197-201 of P450 1A2, respectively. Vector v2 (red cone) connects the centers of the first and last helical turns in helix I defined by centers of the Cα atoms of residues 305-309 and 332-336 of P450 1A2, respectively. Vectors v3 (yellow cone) and v4 (green cone) are placed along P450 1A2 TM helix (residues 7-33) and cyt b5 TM helix (residues 113-130), respectively. Center of mass of the P450 1A2 soluble domain (Cα atoms of residues 34-513), P450 1A2 TM helix (Cα atoms of residues 7-33), and cyt b5 TM helix (Cα atoms of residues 113-130) are named M1 (light blue point), M2 (yellow point) and M3 (green point). P450 1A2 (light grey) and cyt b5 (orange) are shown as a cartoon. Bead representing a phosphate group in DLPC molecules is shown as light grey ball. Heme cofactors of P450 1A2 and cyt b5 are shown as black sticks. Oxygen atoms of heme propionates are shown as red balls and iron atoms as orange balls. Dashed black line shows center of the phospholipid bilayer. Note that the orientation of the cyt b5 soluble domain with respect to the P450 1A2 domain is fixed via the elastic network. Figure 3. Orientation of the TM domain of cyt b5 in the membrane during CG MD simulations (left). Trace of the protein is shown as a dark gray stick. Side chain beads are shown as colored balls; ball colors denote amino acid character (white – nonpolar, green -


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polar, orange – aromatic, blue – positive charge. DLPC molecules are shown as light gray lines; beads representing choline and phosphate groups are shown as light gray balls. Population analysis of the tilt and ζ angle distribution (right); the angle was sampled every 0.2 ns and the size of intervals was 5°. The dashed line and blue strip indicate the adjusted experimental value and experimental error, respectively. Figure 4. Spontaneous association of the cyt b5 TM helix with P450 1A2 TM helix during an unconstrained CG MD of the membrane bound P450 1A2–cyt b5 complex. Soluble domains and polar head groups of the phospholipid bilayer are depicted as gray ellipses and strips, respectively. The initial positions of the P450 1A2 and cyt b5 TM domains are shown as grey and red tubes, respectively. For each subsequent trajectory snapshot, residues 9-29 of the P450 1A2 TM helix were aligned to their initial positions, while depicting the positions of the cyt b5 TM helix at 10 ns intervals by balls that were placed in the middle segment of the TM helix (A120). Varying from red to blue, the ball color indicates the trajectory progress; the final position of the cyt b5 TM helix is represented by the blue tube. The spontaneous association of TM helices is independent of unstructured cyt b5 linker. Figure 5. Rmsd distribution for cyt b5 backbone atoms in the P450 1A2–cyt b5 complex in the absence (black) and presence (green) of the membrane and TM domains. The rmsd reference was the docked structure published previously.24 The inset shows overlap of individual complexes viewed from the top and perpendicularly to the presumed membrane; structures of cyt b5 in modes sAI, sAII and sB are shown in yellow, blue and pink colors, respectively. P450 1A2 is shown as gray ribbon and its heme cofactor as red sticks. Figure 6. Representative structures of the membrane bound P450 1A2–cyt b5 complex in modes mAI and mB obtained from all-atom MD simulations. P450 1A2 (light grey) and cyt b5 (orange) are shown in cartoon representation. Phosphate atoms of lipid molecules are shown as light grey balls. Heme cofactors of P450 1A2 and cyt b5 are shown as black sticks. Oxygen 41 Environment ACS Paragon Plus


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atoms of heme propionates are shown as red balls and iron atoms as orange balls. F helix, F’G’ loop, and G helix are shown in pink and the I helix in green. Figure 7. Percentage occurrence of ionic hydrogen bonds between soluble domains of P450 1A2 and cyt b5 in the soluble (gray) and membrane bound (red) complex. The presence of the salt bridges was monitored using the program VMD using donor-acceptor distance cut-off 3 Å and angle cut-off 40 degrees. Only salt bridges stable for more than 20% of the simulation time are shown. The positions residues forming these salt bridges for both complexes are shown in supporting information (Figure S7 and S8). Figure 8. Spatial distribution of the cyt b5 TM helix in relation to the stationary P450 1A2 TM helix. TM domains of P450 1A2 and cyt b5 that were sampled along an unconstrained allatom MD of the full-length P450 1A2–cyt b5 complex are shown as dark grey cartoons and light gray transparent cylinders. Residues 9 to 29 of the P450 1A2 TM helix were aligned in the each step to the initial structure. (A) Side view; grey circles show positions of phosphate groups of individual phosphatidylcholines and black arrow indicate the view point of the part B. (B) View from the C terminal end of the P450 1A2 TM domain. (C) Population distribution of the angle between TM domains of P450 1A2 and cyt b5 (η) and the distribution of angles γ and ζ between the phospholipid bilayer and TM domains of the P450 1A2 and cyt b5, respectively. Figure 9. Distribution of electron donor−acceptor distances (top) and buried surface areas (bottom) in the soluble (light and dark blue) and membrane-bound (orange and red) P450 1A2–cyt b5 complex. rda was defined as the edge-to-edge distance between redox centers of P450 1A2 and cyt b5. Because the two hemes adopt nearly T-shaped geometry in all studied complexes, this distance was approximated as the Fe–Fe distance minus the sum of the porphyrin disk radius (4.2 Å) and the Fe–S bond distance (2.32 Å). Buried surface area was measured as a difference between the summa of the solvent accessible surface area of the 42 Environment ACS Paragon Plus

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soluble domain of P450 1A2 (residues 34 to 513) and cyt b5 (7 to 92) and the area of the whole complex.



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FIGURES

Figure 1. Schematic strategy for the assembly of the full-length membrane-bound P450 1A2– cyt b5 complex. The P450 1A2 and cyt b5 proteins in CG model are shown in black and blue, respectively. The P450 1A2 and cyt b5 proteins simulated using all-atom MD are shown in magenta and lime, respectively. For a detailed description of individual steps, see Methods.


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Figure 2. Definition of vectors (v1-v4) and centers of mass (M1-M3) describing the orientation of the P450 1A2–cyt b5 complex in the phospholipid bilayer. Vector v1 (blue cone) connects one helical turn in helix C and one in helix F, i.e. the center of the Cα atoms of residues 127131 and 197-201 of P450 1A2, respectively. Vector v2 (red cone) connects the centers of the first and last helical turns in helix I defined by centers of the Cα atoms of residues 305-309 and 332-336 of P450 1A2, respectively. Vectors v3 (yellow cone) and v4 (green cone) are placed along P450 1A2 TM helix (residues 7-33) and cyt b5 TM helix (residues 113-130), respectively. Center of mass of the P450 1A2 soluble domain (Cα atoms of residues 34-513), P450 1A2 TM helix (Cα atoms of residues 7-33), and cyt b5 TM helix (Cα atoms of residues 113-130) are named M1 (light blue point), M2 (yellow point) and M3 (green point). P450 1A2 (light grey) and cyt b5 (orange) are shown as a cartoon. Bead representing a phosphate group in DLPC molecules is shown as light grey ball. Heme cofactors of P450 1A2 and cyt b5 are shown as black sticks. Oxygen atoms of heme propionates are shown as red balls and iron atoms as orange balls. Dashed black line shows center of the phospholipid bilayer. Note that the orientation of the cyt b5 soluble domain with respect to the P450 1A2 domain is fixed via the elastic network.



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Figure 3. Orientation of the TM domain of cyt b5 in the membrane during CG MD simulations (left). Trace of the protein is shown as a dark gray stick. Side chain beads are shown as colored balls; ball colors denote amino acid character (white – nonpolar, green polar, orange – aromatic, blue – positive charge. DLPC molecules are shown as light gray lines; beads representing choline and phosphate groups are shown as light gray balls. Population analysis of the tilt and ζ angle distribution (right); the angle was sampled every 0.2 ns and the size of intervals was 5°. The dashed line and blue strip indicate the adjusted experimental value and experimental error, respectively.


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Figure 4. Spontaneous association of the cyt b5 TM helix with P450 1A2 TM helix during an unconstrained CG MD of the membrane bound P450 1A2–cyt b5 complex. Soluble domains and polar head groups of the phospholipid bilayer are depicted as gray ellipses and strips, respectively. The initial positions of the P450 1A2 and cyt b5 TM domains are shown as grey and red tubes, respectively. For each subsequent trajectory snapshot, residues 9-29 of the P450 1A2 TM helix were aligned to their initial positions, while depicting the positions of the cyt b5 TM helix at 10 ns intervals by balls that were placed in the middle segment of the TM helix (A120). Varying from red to blue, the ball color indicates the trajectory progress; the final position of the cyt b5 TM helix is represented by the blue tube. The spontaneous association of TM helices is independent of unstructured cyt b5 linker.



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Figure 5. Rmsd distribution for cyt b5 backbone atoms in the P450 1A2–cyt b5 complex in the absence (black) and presence (green) of the membrane and TM domains. The rmsd reference was the docked structure published previously.24 The inset shows overlap of individual complexes viewed from the top and perpendicularly to the presumed membrane; structures of cyt b5 in modes sAI, sAII and sB are shown in yellow, blue and pink colors, respectively. P450 1A2 is shown as gray ribbon and its heme cofactor as red sticks.


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Figure 6. Representative structures of the membrane bound P450 1A2–cyt b5 complex in modes mAI and mB obtained from all-atom MD simulations. P450 1A2 (light grey) and cyt b5 (orange) are shown in cartoon representation. Phosphate atoms of lipid molecules are shown as light grey balls. Heme cofactors of P450 1A2 and cyt b5 are shown as black sticks. Oxygen atoms of heme propionates are shown as red balls and iron atoms as orange balls. F helix, F’G’ loop, and G helix are shown in pink and the I helix in green.



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Figure 7. Percentage occurrence of ionic hydrogen bonds between soluble domains of P450 1A2 and cyt b5 in the soluble (gray) and membrane bound (red) complex. The presence of the salt bridges was monitored using the program VMD using donor-acceptor distance cut-off 3 Å and angle cut-off 40 degrees. Only salt bridges stable for more than 20% of the simulation time are shown. The positions residues forming these salt bridges for both complexes are shown in supporting information (Figure S7 and S8).


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Figure 8. Spatial distribution of the cyt b5 TM helix in relation to the stationary P450 1A2 TM helix. TM domains of P450 1A2 and cyt b5 that were sampled along an unconstrained allatom MD of the full-length P450 1A2–cyt b5 complex are shown as dark grey cartoons and light gray transparent cylinders. Residues 9 to 29 of the P450 1A2 TM helix were aligned in the each step to the initial structure. (A) Side view; grey circles show positions of phosphate groups of individual phosphatidylcholines and black arrow indicate the view point of the part B. (B) View from the C terminal end of the P450 1A2 TM domain. (C) Population distribution of the angle between TM domains of P450 1A2 and cyt b5 (η) and the distribution of angles γ and ζ between the phospholipid bilayer and TM domains of the P450 1A2 and cyt b5, respectively.



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Figure 9. Distribution of electron donor−acceptor distances (top) and buried surface areas (bottom) in the soluble (light and dark blue) and membrane-bound (orange and red) P450 1A2–cyt b5 complex. rda was defined as the edge-to-edge distance between redox centers of P450 1A2 and cyt b5. Because the two hemes adopt nearly T-shaped geometry in all studied complexes, this distance was approximated as the Fe–Fe distance minus the sum of the porphyrin disk radius (4.2 Å) and the Fe–S bond distance (2.32 Å). Buried surface area was measured as a difference between the summa of the solvent accessible surface area of the soluble domain of P450 1A2 (residues 34 to 513) and cyt b5 (7 to 92) and the area of the whole complex.


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Membrane-Anchored Cytochrome P450 1A2âCytochrome b5

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