A 175-psec molecular dynamics simulation of camphor-bound cytochrome P-450cam.

The structure and internal motions of cytochrome P-450cam, a monooxygenase heme enzyme with 414 amino acid residues, with camphor bound at the active site have been evaluated on the basis of a 175-psec molecular dynamics simulation carried out at 300 K. All hydrogen atoms were explicitly modeled, and 204 crystallographic waters were included in the simulation. Based on an analysis of the time course of the trajectory versus potential energy, root mean square deviation, radius of gyration, and hydrogen bonding, the simulation was judged to be stable and representative of the average experimental structure. The averaged structural properties of the enzyme were evaluated from the final 135 psec of the simulation. The average atomic displacement from the X-ray structure was 1.39 A for all heavy atoms and 1.17 A for just C-alpha atoms. The average root-mean-square (rms) fluctuations of all heavy atoms and backbone atoms were 0.42 and 0.37 A, respectively. The computed rms fluctuations were in reasonable agreement with the experimentally determined temperature factors. All 13 segments of alpha-helix and 5 segments of beta-sheet were well preserved with the exception of the N-terminal half of helix F which alternated between an alpha-helix and a 310-helix. In addition there were in general only small variations in the relative orientation of adjacent alpha-helices. The rms fluctuations of the backbone dihedral angles in the secondary structure elements were almost uniformly smaller, with the fluctuation in alpha-helices and beta-sheets, 31 and 10% less, respectively, than those in nonsecondary structure regions. The reported crystal structure contains kinks in both helices C and I. In the simulation, both of these regions showed high mobility and large deviations from their starting positions. Since the kink in the I helix is at the oxygen binding site, these motions may have mechanistic implications.     

Spring-loading the active site of cytochrome P450cam

A hydrogen bond network has been identified that adjusts protein-substrate contacts in cytochrome P450(cam) (CYP101A1). Replacing the native substrate camphor with adamantanone or norcamphor causes perturbations in NMR-detected NH correlations assigned to the network, which includes portions of a β sheet and an adjacent helix that is remote from the active site. A mutation in this helix reduces enzyme efficiency and perturbs the extent of substrate-induced spin state changes at the haem iron that accompany substrate binding. In turn, the magnitude of the spin state changes induced by alternate substrate binding parallel the NMR-detected perturbations observed near the haem in the enzyme active site.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

Thermodynamics of water mediating protein-ligand interactions in cytochrome P450cam: a molecular dynamics study.

Ordered water molecules are observed by crystallography and nuclear magnetic resonance to mediate protein-ligand interactions. Here, we examine the energetics of hydrating cavities formed at protein-ligand interfaces using molecular dynamics simulations. The free energies of hydrating two cavities in the active site of two liganded complexes of cytochrome P450cam were calculated by multiconfigurational thermodynamic integration. The complex of cytochrome P450cam with 2-phenyl-imidazole contains a crystallographically well defined water molecule mediating hydrogen bonds between the protein and the inhibitor. We calculate that this water molecule is stabilized by a binding free energy of -11.6 +/- kJ/mol. The complex of cytochrome P450cam with its natural substrate, camphor, contains a cavity that is empty in the crystal structure although a water molecule in it could make a hydrogen bond to camphor. Here, solvation of this cavity is calculated to be unfavorable by +15.8 +/- 5.0 kJ/mol. The molecular dynamics simulations can thus distinguish a hydrated interfacial cavity from an empty one. They also provide support for the notion that protein-ligand complexes can accommodate empty interfacial cavities and that such cavities are likely to be unhydrated unless more than one hydrogen bond can be made to a water molecule in the cavity.     

CO migration pathways in cytochrome P450cam studied by molecular dynamics simulations

Previous laser flash photolysis investigations between 100 and 300 K have shown that the kinetics of CO rebinding with cytochrome P450(cam)(camphor) consist of up to four different processes revealing a complex internal dynamics after ligand dissociation. In the present work, molecular dynamics simulations were undertaken on the ternary complex P450(cam)(cam)(CO) to explore the CO migration pathways, monitor the internal cavities of the protein, and localize the CO docking sites. One trajectory of 1 nsec with the protein in a water box and 36 trajectories of 1 nsec in the vacuum were calculated. In each trajectory, the protein contained only one CO ligand on which no constraints were applied. The simulations were performed at 200, 300, and 320 K. The results indicate the presence of seven CO docking sites, mainly hydrophobic, located in the same moiety of the protein. Two of them coincide with xenon binding sites identified by crystallography. The protein matrix exhibits eight persistent internal cavities, four of which corresponding to the ligand docking sites. In addition, it was observed that water molecules entering the protein were mainly attracted into the polar pockets, far away from the CO docking sites. Finally, the identified CO migration pathways provide a consistent interpretation of the experimental rebinding kinetics.     

Substrate mobility in a deeply buried active site: analysis of norcamphor bound to cytochrome P-450cam as determined by a 201-psec molecular dynamics simulation.

While cytochrome P-450cam catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor with 100% stereospecificity, norcamphor is hydroxylated by this enzyme yielding 45% 5-exo-, 47% 6-exo-, and 8% 3-exo-hydroxynorcamphor (Atkins, W.M., Sligar, S.G., J. Am. Chem. Soc. 109:3754-3760, 1987). The present study describes a 201-psec molecular dynamics (MD) stimulation of norcamphorbound cytochrome P-450cam to elucidate the relationship between substrate conformational mobility and formation of alternative products. First, these data suggest that the product specificity is, at least partially, due to the mobility of the substrate within the active site. Second, the high mobility of norcamphor in the active site leads to an average increase in separation between the heme iron and the substrate of about 1.0 A; this increase in separation may be the cause of the uncoupling of electron transfer when norcamphor is the substrate. Third, the active site water located in the norcamphorbound crystal structure possesses mobility that correlates well with the spin-state equilibrium of this enzyme-substrate complex.     

Model of the coordination site for cobalt-substituted cytochrome P450cam

Cobalt-substituted cytochrome P450_cam was recently reconstituted by Wagner et al (J. Biol. Chem. 256, 6266, (1981)). A model of its coordination site was constructed to determine the mode of axial coordination of the native enzyme. Complexes were prepared from cobalt porphyrins (cobalt-protoporphyrin IX (CoPPIX), cobalt-meso-tetraphenylporphyrin, cobalt-γ-laurylpyridyl triphenylporphyrin, and cobalt-octaethylporphyrin), thioglycolate ester, and tetramethylammonium hydroxide in organic solvents. Complexes prepared in an organic solvent such as CHC1₃ under air at room temperature exhibited a stable Soret hyperporphyrin spectrum characterized by split Soret bands, especially like that of the thiol-Co-P450_cam complex Comparison of the spectra of the hyperporphyrin spectral complexes titrated with various types of alcohol and imidazole, with the spectrum of Co-P450_cam in the oxidized state support the idea that an axial thiolate at the fifth position and a hydroxyl group of alcohol at the sixth position of the heme form the coordination site of Co-P450_cam The CoPPIX-thiolate-ethanol complex retaining S⁻-Co(III)-OH coordination is thought to be a possible model of Co-P450_cam in the oxidized state.     

Experimentally restrained molecular dynamics simulations for characterizing the open states of cytochrome P450cam

Residual dipolar couplings (RDCs) were used as restraints in fully solvated molecular dynamics simulations of reduced substrate- and carbonmonoxy-bound cytochrome P450(cam) (CYP101A1), a 414-residue soluble monomeric heme-containing camphor monooxygenase from the soil bacterium Pseudomonas putida. The (1)D(NH) residual dipolar couplings used as restraints were measured in two independent alignment media. A soft annealing protocol was used to heat the starting structures while incorporating the RDC restraints. After production dynamics, structures with the lowest total violation energies for RDC restraints were extracted to identify ensembles of conformers accessible to the enzyme in solution. The simulations result in substrate orientations different from that seen in crystallographic structures and a more open and accessible enzyme active site and largely support previously reported differences between the open and closed states of CYP101A1.     

Spectroelectrochemistry of cytochrome P450cam

The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis.     

Progress in cytochrome P450 active site modeling

Models capable of predicting the possible involvement of cytochromes P450 in the metabolism of drugs or drug candidates are important tools in drug discovery and development. Ideally, functional information would be obtained from crystal structures of all the cytochromes P450 of interest. Initially, only crystal structures of distantly related bacterial cytochromes P450 were available-comparative modeling techniques were used to bridge the gap and produce structural models of human cytochromes P450, and thereby obtain some useful functional information. A significant step forward in the reliability of these models came four years ago with the first crystal structure of a mammalian cytochrome P450, rabbit CYP2C5, followed by the structures of two human enzymes, CYP2C8 and CYP2C9, and a second rabbit enzyme, CYP2B4. The evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism, is presented as a case study.     

Ethylbenzene hydroxylation by cytochrome P450cam.

The metabolism of ethylbenzene by cytochrome P450cam was analyzed by experimental and theoretical methods. The present experiments indicate that ethylbenzene is hydroxylated almost exclusively at the secondary ethyl carbon with about a 2:1 ratio of R:S product. Several molecular dynamics trajectories were performed with different starting conformations of ethylbenzene in the active site of P450cam. The stereochemistry of hydroxylation predicted from the molecular dynamics simulations was found to be in good agreement with the observed products.     

Molecular dynamics simulations of phenylimidazole inhibitor complexes of cytochrome P450 cam

Molecular dynamics simulations have been performed on three phenylimidazole inhibitor complexes ofP450 _cam, utilizing the X-ray structures and the AMBER suite of programs. Compared to their corresponding optimized X-ray structures, very similar features were observed for the 1-phenylimidazole (1-PI) and 2-phenylimidazole (2-PI) complexes during a 100 ps MD simulation. The 1-PI inhibitor binds as a Type II complex with the imidazole nitrogen as a ligand of the heme iron. Analysis of the inhibitor-enzyme interctions during the MD simulations reveals that electrostatic interactions of the imidazole with the heme and van der Waals interactions of the phenyl ring with nearby hydrophobic residues are dominant. By contrast, 2-PI binds as a Type I inhibitor in the substrate binding pocket, but not as a ligand of the iron. The interactions of this inhibitor are qualitatively different from that of the Type II 1-PI, being mainly electrostatic/H-bonding interactions with a bound water and polar residues. Although the third compound, 4-PI, in common with 1-PI, also binds as a Type II inhibitor, with one nitrogen of the imidazole as a ligand to the iron, the MD average binding orientation deviates significantly from the X-ray structure. The most important changes observed include: (1) the rotation of the imidazole ring of this inhibitor by about 90° to enhance electrostatic interactions of the imidazole NH group with the carbonyl group of LEU244, and (2) the rotation of the carbonyl group of ASP251 to form a H-bond with VAL254. An analysis of the H-bonding network surrounding this substrate in the optimized crystal structure revealed that there is no H-bonding partner either for the free polar NH group in the imidazole ring of 4-phenylimidazole or for the polar carbonyl group of the nearby ASP251 residue. The deviation of the dynamically averaged inhibitor-enzyme structure of the 4-PI complex from the optimized crystal structure can therefore be rationalized as a consequence of the optimization of the electrostatic interactions among the polar groups.     

Molecular dynamics study of time-correlated protein domain motions and molecular flexibility: cytochrome P450BM-3.

Time-correlated atomic motions were used to characterize protein domain boundaries from atomic coordinates generated by molecular dynamics simulations. A novel application of the dynamical cross-correlation matrix (DCCM) analysis tool was used to help identify putative protein domains. In implementing this new approach, several DCCM maps were calculated, each using a different coordinate reference frame from which protein domain boundaries and protein domain residue constituents could be identified. Cytochrome P450BM-3, from Bacillus megaterium, was used as the model protein in this study. The analyses indicated that the simulated protein comprises three distinct domain regions; in contrast, only two protein domains were identified in the original crystal structure report. Specifically, the DCCM analyses showed that the F-G helix region was a separate domain entity and not a part of the alpha domain, as previously designated. The simulations demonstrated that the domain motions of the F-G helix region effected both the size and shape of the enzyme active site, and that the dynamics of the F-G helix domain could possibly control access of substrate to the binding pocket.     

High-resolution crystal structure of cytochrome P450cam

The crystal structure of Pseudomonas putida cytochrome P450cam with its substrate, camphor, bound has been refined to R = 0.19 at a normal resolution of 1.63 A. While the 1.63 A model confirms our initial analysis based on the 2.6 A model, the higher resolution structure has revealed important new details. These include a more precise assignment of sequence to secondary structure, the identification of three cis-proline residues, and a more detailed picture of substrate-protein interactions. In addition, 204 ordered solvent molecules have been found, one of which appears to be a cation. The cation stabilizes an unfavorable polypeptide conformation involved in forming part of the active site pocket, suggesting that the cation may be the metal ion binding site associated with the well-known ability of metal ions to enhance formation of the enzyme-substrate complex. Another unusual polypeptide conformation forms the proposed oxygen-binding pocket. A localized distortion and widening of the distal helix provides a pocket for molecular oxygen. An intricate system of side-chain to backbone hydrogen bonds aids in stabilizing the required local disruption in helical geometry. Sequence homologies strongly suggest a common oxygen-binding pocket in all P450 species. Further sequence comparisons between P450 species indicate common three-dimensional structures with changes focused in a region of the molecule postulated to be associated with the control of substrate specificity.     

Structural evidence for a functionally relevant second camphor binding site in P450cam: model for substrate entry into a P450 active site

P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F-G helix that covers the active site pocket. Such a two-step binding process is kinetically required if P450cam rarely populates an open conformation-as suggested by previous literature and the inability to obtain a crystal structure of P450cam in an open conformation. Such a mechanism would minimize exposure of the heme by allowing P450cam to stay in a closed conformation as long as possible, since only brief flexing into an open conformation would be required to allow substrate entry. To test this model, we have attempted to dock a second camphor molecule into the crystal structure of camphor-bound P450cam. The docking identified only one potential entry site pocket, a well-defined cavity on the F-helix side of the F-G flap, 16 A from the heme iron. Location of this entry site pocket is consistent with our NMR T1 relaxation-based measurements of distances for a camphor that binds in fast exchange (active site camphor is known to bind in slow exchange). Presence of a second camphor binding site is also confirmed with [(1)H-(13)C] HSQC titrations of (13)CH3-threonine labeled P450cam. To confirm that camphor can bind outside of the active site pocket, (13)CH3-S-pyridine was bound to the heme iron to physically block the active site, and to serve as an NMR chemical shift probe. Titration of this P450cam-pyridine complex confirms that camphor can bind to a site outside the active site pocket, with an estimated Kd of 43 microM. The two-site binding model that is proposed based on these data is analogous to that recently proposed for CYP3A4, and is consistent with recent crystal structures of P450cam bound to tethered-substrates, which force a partially opened conformation.     

Conformational states of cytochrome P450cam revealed by trapping of synthetic molecular wires

Members of the ubiquitous cytochrome P450 family catalyze a vast range of biologically significant reactions in mammals, plants, fungi, and bacteria. Some P450s display a remarkable promiscuity in substrate recognition, while others are very specific with respect to substrate binding or regio and stereo-selective catalysis. Recent results have suggested that conformational flexibility in the substrate access channel of many P450s may play an important role in controlling these effects. Here, we report the X-ray crystal structures at 1.8A and 1.5A of cytochrome P450cam complexed with two synthetic molecular wires, D-4-Ad and D-8-Ad, consisting of a dansyl fluorophore linked to an adamantyl substrate analog via an alpha,omega-diaminoalkane chain of varying length. Both wires bind with the adamantyl moiety in similar positions at the camphor-binding site. However, each wire induces a distinct conformational response in the protein that differs from the camphor-bound structure. The changes involve significant movements of the F, G, and I helices, allowing the substrate access channel to adapt to the variable length of the probe. Wire-induced opening of the substrate channel also alters the I helix bulge and Thr252 at the active site with binding of water that has been proposed to assist in peroxy bond cleavage. The structures suggest that the coupling of substrate-induced conformational changes to active-site residues may be different in P450cam and recently described mammalian P450 structures. The wire-induced changes may be representative of the conformational intermediates that must exist transiently during substrate entry and product egress, providing a view of how substrates enter the deeply buried active site. They also support observed examples of conformational plasticity that are believed be responsible for the promiscuity of drug metabolizing P450s. Observation of such large changes in P450cam suggests that substrate channel plasticity is a general property inherent to all P450 structures.     

Dynamics underlying hydroxylation selectivity of cytochrome P450cam

Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.     

Enantiomeric discrimination of Ru-substrates by cytochrome P450cam

Molecules with photosensitizers attached to substrates (Wilker et al., Angew. Chem. Int. Ed. 38 (1999) 90-92) or cofactors (Hamachi et al., J. Am. Chem. Soc. 121 (1999) 5500-5506) can rapidly deliver redox equivalents to buried active sites. The structure of cytochrome P450cam (P450) co-crystallized with a prototypal sensitizer-substrate, [Ru-C9-Ad]Cl2, has been determined (Dmochowski et al., Proc. Natl. Acad. Sci. USA 96 (1999) 12987-12990); and, in separate UV-vis absorption and time-resolved luminescence experiments, the binding of the lambda and delta enantiomers of Ru-C9-Ad to P450 has been measured. The results, KD(delta/lambda) approximately 2, indicate that the bipyridyl ligands of the lambda isomer interact more favorably with hydrophobic residues at the entrance to the substrate channel. We conclude that enantiospecific interactions may be exploited in the design of enzyme-metallosubstrate conjugates.     

Topological role of cytochrome P450 2D6 active site residues

Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.     

Ketoconazole-induced conformational changes in the active site of cytochrome P450eryF

The azole-based P450 inhibitor ketoconazole is used to treat fungal infections and functions by blocking ergosterol biosynthesis in yeast. Ketoconazole binds to mammalian P450 enzymes and this can result in drug-drug interactions and lead to liver damage. To identify protein-drug interactions that contribute to binding specificity and affinity, we determined the crystal structure of ketoconazole complexed with P450eryF. In the P450eryF/ketoconazole structure, the azole moiety and nearby rings of ketoconzole are positioned in the active site similar to the substrate, 6-deoxyerythronolide B, with the azole nitrogen atom coordinated to the heme iron atom. The remainder of the ketoconazole molecule extends into the active-site pocket, which is occupied by water in the substrate complex. Binding of ketoconazole led to unexpected conformational changes in the I-helix. The I-helix cleft near the active site has collapsed with a helical pitch of 5.4 A compared to 6.6 A in the substrate complex. P450eryF/ketoconazole crystals soaked in 6-deoxyerythronolide B to exchange ligands exhibit a structure identical with that of the original P450eryF/substrate complex, with the I-helix cleft restored to a pitch of 6.6 A. These findings indicate that the I-helix region of P450eryF is flexible and can adopt multiple conformations. An improved understanding of the flexibility of the active-site region of cytochrome P450 enzymes is important to gain insight into determinants of ligand binding/specificity as well as to evaluate models for catalytic mechanism based on static crystal structures.