Peroxidase Site of Prostaglandin Endoperoxide H Synthase-1

May 28, 2004 - Dynamics Studies with a Prostaglandin Endoperoxide Analog ... Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and PGHS-2) ...
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J. Phys. Chem. B 2004, 108, 9297-9305

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Peroxidase Site of Prostaglandin Endoperoxide H Synthase-1: Docking and Molecular Dynamics Studies with a Prostaglandin Endoperoxide Analog Steve A. Seibold,† William L. Smith,§ and Robert I. Cukier*,‡ Department of Chemistry and the Center for Biological Modeling, Michigan State UniVersity, East Lansing, Michigan 48823, and Department of Biological Chemistry, UniVersity of Michigan Medical School, Ann Arbor, Michigan 48109 ReceiVed: January 12, 2004; In Final Form: April 19, 2004

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and PGHS-2) catalyze the first step in the biosynthetic pathway that produces prostaglandins and thromboxanes. The fatty acid endoperoxide/ hydroperoxide substrate, PGG2, binds on the distal side of the heme that forms the peroxidase (POX) site of PGHSs and generates the alcohol PGH2 by cleaving the oxygen-oxygen bond of the 15-hydroperoxide group. The structure of the POX site of PGHS, as with other peroxidases, includes the invariant distal histidine residue His207 and a glutamine, Gln203. We report the first molecular dynamics (MD) simulation of a PGG2 analogue (pseudo-PGG2) bound to the peroxidase site of PGHS-1; pseudo-PGG2 lacks the endoperoxide group and double bonds of PGG2 but is otherwise identical to PGG2. In the MD of the substrate-free state of PGHS1, a water migrated to the heme active site to become the sixth ligand of the iron, and in time, it hydrogen bonded to other waters, forming chains that extended into the bulk solvent. A location for pseudo-PGG2 was found by scanning for low van der Waals contact energies in the distal heme pocket, and its geometry was refined by simulated annealing, but the resulting position was still not close enough to the iron or His207 to support catalysis. During a 2-ns MD simulation of the complex, the hydroperoxide oxygens move to within hydrogen-bonding distance of His207 and Gln203 and ligate to the heme iron, positioning the substrate appropriately for peroxidase catalysis. Water is excluded from the active site by this large substrate.

Introduction Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and PGHS-2) catalyze the first step in the biosynthetic pathway that produces prostaglandins from the substrate arachidonic acid. PGHS-1 and -2 are isozymes with about 60% sequence identity. The enzymes are encoded by separate genes that are expressed differentially.1 The work described here will be specific to ovine PGHS-1, and for convenience, we henceforth designate it as PGHS. It has two catalytic sites. One, the cyclooxygenase (COX) site, catalyzes the bis-oxygenation of arachidonic acid to produce the 15-hydroperoxy-9,11-endoperoxide prostaglandin G2 (PGG2). The other, the peroxidase (POX) site, performs the two-electron reduction of the 15-hydroperoxyl group of PGG2 to form PGH2, the corresponding alcohol. (See Scheme 1.)2,3 The structure of the POX site of PGHS is similar to that of other peroxidases in that it contains an invariant distal histidine (His207) and a nearby residue that may also support hydrogen bonding; Gln203 in the case of PGHS. (See Figure 1.) PGHS produces the typical peroxide intermediate species found in other heme peroxidases.4,5 The POX activity involves the binding of the 15-hydroperoxyl group of PGG2 to the heme iron with the concomitant donation of a proton to the distal His207 from the R (terminal)-oxygen of the peroxyl group coordinated to the heme iron. Subsequently, there is transfer of a proton from His207 to the β oxygen that * Corresponding author. Fax: (517)-353-1793. Tel: (517)-355-9715, ext. 263. E-mail: [email protected]. † Department of Chemistry, Michigan State University. ‡ Center for Biological Modeling, Michigan State University. § University of Michigan Medical School.

results in an acid-base-catalyzed heterolytic cleavage of the oxygen-oxygen bond. This yields PGH2 and a peroxidase, compound I, a species having an oxoferryl group and a heme radical cation.6 Compound I can undergo a one-electron reduction by an intramolecular electron donor to form intermediate II consisting of a tyrosyl radical and the oxoferryl heme. The Tyr385 radical is the species that abstracts the 13proS hydrogen from arachidonic acid to initiate the COX activity.1,3,7,8 The push-pull mechanism is often used to explain peroxidase catalysis. The distal histidine and glutamine (in the case of PGHS) supply the pull by hydrogen bonding to the substrate, and the proximal histidine that is ligated to the heme iron, acting as a Lewis base, purportedly provides the push.9,10 However, there are several reports of peroxidases, including PGHS,11 where the push appears to be weak or nonexistent. To make the pull part of the mechanism feasible, the distal histidine (His207) and glutamine (Gln203) must be in position to interact with the peroxide group of the substrate. In addition, the peroxide group must be close to the heme iron. The amino acids comprising both the heme binding and the peroxidase active site are part of a helix bundle3 whose structure is conserved in the related enzymes, such as myeloperoxidase.12,13 However, the preferred substrate of myeloperoxidase is hydrogen peroxide,14 whereas PGHS uses preferentially secondary alkyl hydroperoxides such as the large substrate PGG2.15 The goal of the work reported here was to develop a method to dock a large substrate comparable to PGG2 into the peroxidase active site of PGHS and then to perform molecular dynamics (MD) on the substrate-protein complex. The ligand we choose will be referred to as pseudo-PGG2. It is missing the endoperoxide oxygens and has single bonds where PGG2 has double

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SCHEME 1: COX Site of PGHS Oxygenates AA by the Incorporation of Molecular Oxygen to Produce PGG2, Containing the Endoperoxide and the Hydroperoxide Groupsa

Figure 1. Peroxidase active site of ovine PGHS-1 based on the X-ray crystal structure (1CQE). The plane of the heme separates a distal region (above the plane) that is the binding site for hydroperoxide substrates and a proximal region (below the plane) containing the iron-ligated histidine. Some key residues for peroxidase and cyclooxygenase catalysis are also shown and labeled, as are the propionates.

Methods

a At a physically separate location, the POX site of the enzyme, PGG2 is reduced by two electron equivalents to form the PGH2 alcohol.

bonds. Its dimensions and structure (a seven-carbon chain terminating with a carboxylate tail, a five-membered-ring headgroup, and an eight-carbon chain containing the 15S hydroperoxide group) are otherwise identical to those of PGG2. (See Scheme 1.) Because of the size and flexibility of this substrate, the protein-substrate system has multiple conformers; hence, a starting configuration of sufficiently low energy is difficult to generate. A program was written to determine the available space for a large substrate that would not lead to severe van der Waals clashes with the protein. Subsequent use of a Monte Carlo-based simulated annealing method with a protocol designed to fine tune the docking produced a best-fit docked substrate. Once suitably docked, MD was performed to determine if pseudo-PGG2 would form a catalytically competent complex with the protein. The appropriateness for catalysis of the MD-generated complex was inferred from the requirements for hydrogen bonding of distal His207 and Gln203 with the substrate and the proximity (ca. 2.2 Å) of the peroxide oxygens to the heme iron. These are minimal conditions for a pushpull mechanism with PGHS.

Docking Studies. Ligands were constructed and docked into the POX site of the crystallographic PGHS structure (1CQE12) with the use of the Molecular Operating Environment (MOE) package.16 The charges on the ligand and the protein were obtained from the CHARMm22 17 parameter set. The ligands were docked into the POX binding site using the AUTODOCK docking program, part of the MOE suite. This MOE-Dock program incorporates both manual and automatic docking procedures. The docking routine evaluates both nonbonded van der Waals and electrostatic interactions. This ligand docking procedure, which allows for flexibility of the ligand but not the protein, was then used to obtain the position of the lowest-energy conformation for each ligand in the POX site using a Monte Carlo (simulated annealing)-based automated protocol. To locate possibilities for the position and orientation of a large substrate that do not lead to prohibitively large van der Waals interactions with the protein, we developed a simple search algorithm. A van der Waals probe of 1.4-Å radius is scanned on a cubic grid with 1-Å grid spacing. The interaction energy of the probe with the protein is evaluated at each grid point and recorded if it is less than zero. A picture of this procedure applied to PGHS where the search volume of linear dimension 10 Å that is centered on the heme iron is displayed in Figure 2a. The positions of the spheres show available space in PGHS for possible substrate binding. The spheres have been connected by bars as an aid in gaining a 3D perspective. Pseudo-PGG2 and Pseudo-PGG2-PGHS Complex. The pseudo-PGG2 substrate was generated in MOE and energy minimized using the CHARMm22 17 atom types in the gas phase. The latter coordinates were then used to generate a Gaussian18 input file for geometry optimization, which was carried out by the Hartree-Fock method using a 6-31g** basis set. Atom-centered charges of the electrostatic part of the force field for MD were then generated with the Merz-Kollman method.18 Pseudo-PGG2 was docked to PGHS by using a modified simulated annealing protocol. (See the Results section.) The coordinates of the best-docked pseudo-PGG2-PGHS complex were then moved to LEaP,19 where a topology and new coordinate file were made for the incorporation into SANDER for the MD simulations.

Docking/Molecular Dynamics of a PG Analog

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Figure 2. (a) Available space for hydroperoxide substrate docking in a cubic volume centered on the heme iron of PGHS. The spheres are centered at lattice points where the van der Waals energy of interaction of a 1.4-Å test probe with the protein is small. The bars connecting the spheres are to help visualize the space. The picture is of the distal region, viewed from above the heme plane with the propionates oriented to the left in the direction of the bulk solvent. Phe409 is labeled to show that there is accessible space for ligand docking underneath this residue, which led toward a productive binding in our simulated annealing studies. (b) Available space for peroxide substrate docking in a cubic volume centered on the heme iron myeloperoxidase (MPO). See the legend to part a for details.

Molecular Dynamics. The starting structure of PGHS (using 1CQE or 1EBV20) with ∼15 000 explicit water molecules in a box of approximate dimensions 90 × 90 × 90 Å3 with all crystallographic waters removed was generated in AMBER using the LEaP module. The temperature was kept constant at 300 K with a Berendsen thermostat.21 The particle-mesh Ewald method22 was used to treat the long-range Coulombic interactions with the default values of the SANDER23 program. The ionization states of all residues were set to pH 7. All of the histidines were assumed to be neutral with ND atoms protonated. The proximal histidine, His388, was covalently linked to the heme Fe3+ through the NE nitrogen. The SHAKE algorithm was used to constrain bond lengths involving hydrogens, permitting a time step of 2 fs. MD was performed with the SANDER module of the AMBER package.23 Atom types and parameters for the heme developed by D. A. Giammona as modified by C. Bayly were employed.24 The oxidation state of heme b was set to ferric, and with the assumption that the propionates are ionized, the total charge of the protein is zero. It should be noted that both 1CQE and 1EBV gave similar results; hence, no distinction will be made between them throughout the text. The protein and water were first run at constant NPT for 6 ps to adjust the density to ∼1 g/cc and then switched to the NVT ensemble for the duration of an ∼1-ns simulation. In the case of the pseudo-PGG2-PGHS complex, the total charge is -1 because of the ionized carboxylate tail of the substrate. The system was neutralized by the addition of a sodium ion. Some care in the placement of the ion was required; SANDER placed the ion near the carboxylate group of pseudoPGG2. Preliminary simulations showed that this interaction was maintained and influenced the position of the substrate. Therefore, the ion was transferred to a region of the solvent remote from the carboxylate for the simulations reported here. The system was also allowed to reach a density of ∼1 g/cc and was then switched to the NVT ensemble. The solvent was allowed to relax further during a decrease of the protein restraints from 100 to 0 kcal/mol/Å2 over a 100-ps time interval. Then, the simulation was run for 2 ns.

Figure 3. Distance between the oxygen of the root water molecule and the iron of the heme versus time. The initial position of the water molecule is >12 Å from the iron but moves to within 2.0 Å by 100 ps, where it remains throughout the simulation.

Results Molecular Dynamics of PGHS. MD on PGHS without a ligand was carried out to make sure that the protein was stable during the simulation. Over the 1-ns MD run, the overall main chain rms deviation from the X-ray structure was 1.9 Å. An examination of the rms deviations on a per residue basis produced a conventional pattern where residues in the protein’s interior show rms values on the order of 1.5 Å. Most of the larger rms values are for solvent exposed residues. Another reason to simulate the substrate-free state is the evidence from Raman spectroscopy that the heme Fe3+ is six coordinate, with the sixth ligand being water.11 As shown in Figure 3, a water molecule migrates from its initial position, ∼12 Å away from the heme iron, to within 2 Å of the iron and coordinates at the sixth position for the remainder of the simulation. This root water hydrogen bonds to other waters to

9300 J. Phys. Chem. B, Vol. 108, No. 26, 2004 form a chain consisting of four to five waters (counting the root water) that stretches from the heme iron to the bulk solvent. Water chains persist throughout the simulation; however, except for the root water, all of the water molecules in the distal heme water chain were dynamic and exchanged with the bulk solvent. The slowest rate of exchange was found for the water that hydrogen bonded directly to the root water and exhibited a lifetime of >900 ps before drifting away from the heme pocket (data not shown). It is reassuring that water becomes the sixth ligand of the heme iron and that hydrogen-bonded chains form that extend to the bulk solvent because these results mirror our earlier MD simulation that was carried out with CUKMODY, a different MD program that uses a different force field (GROMOS).25 During the simulation, waters in the distal pocket also interacted with side chains of amino acids in the distal pocket. Moreover, at times, these interactions were part of the chains connecting the heme iron with the bulk solvent. Hydrogen bonding between His207 and the root water was consistent throughout the simulation. More transient hydrogen bonding was found between water and Gln203 and the backbone oxygens of Val291 and Leu294, with lifetimes of approximately 200 ps. The carboxylate oxygens of the A-ring propionate of the heme interact with bulk water but, at times, also participate in chains of waters that bridge the heme plane waters to those of the bulk solvent. In contrast, the carboxylate oxygens of the D-ring propionate hydrogen bond with Thr212 (side-chain hydroxyl group and backbone nitrogen) and the ND (side chain NH2) of Gln382 for the duration of the simulation.26 Several nonpolar distal side chains lining the heme pocket that are exposed to the bulk solvent migrate from their crystallographic positions, after the initial water relaxation, when the restraints on the protein were released. The side chain of Leu294 moved from the X-ray structure distance of 11.4 Å from the heme iron to >15 Å in 50 ps and remained approximately at this distance throughout the simulation. The side chain of Leu295 also moved from its crystallographic position of ∼8 Å away from the heme iron to >10 Å by the end of the simulation. Substrate Fragments and Their Docking. In our initial attempts to locate the native docking site of PGG2 in the distal heme pocket, we tried to dock the endoperoxide headgroup of the PGG2 molecule. After evaluating a multitude of configurations, we found that many drastically different docked conformers, far from the distal pocket interior, had similar electrostatic and van der Waals energies. In another attempt, a three-carbon unit containing the peroxide group, CH3CH2CH2OOH, was constructed and used as the docking fragment. Some of the docked configurations of this ligand allowed the approach of the peroxide group to the heme iron in the distal pocket but led to high van der Waals energies. In fact, the most favorable docking energy between the protein and this ligand resulted in what would be a catalytically inactive species, with large distances between the peroxide group and the heme iron. Thus, two problems arose in these fragment docking attempts: (a) a lack of specificity as exemplified by energetically favorable but catalytically incompetent structures and (b) excessive van der Waals energetics permitting methods such as simulated annealing to succeed. Therefore, we adopted the strategy described next. Docking Protocol and Pseudo-PGG2. To suggest possible locations and orientations of a substrate, we used the van der Waals sphere-scan program described in the Methods section. The result of the scan, centered on the heme iron, is displayed in Figure 2a. For comparison, the same scan is displayed in Figure 2b for myeloperoxidase (MPO),13 a peroxidase that

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Figure 4. Docked structure of pseudo-PGG2. The peroxide group contains the R (terminal) and the β oxygens. The pseudosubstrate is directly analogous to PGG2 in that it contains both the carboxylate and ω-carbon tail; however, it does not contain the endoperoxide oxygens and substitutes carbon-carbon single bonds for the two double bonds.

prefers hydrogen peroxide as its substrate.14 Clearly, the available space for a substrate in PGHS is much greater than in MPO. In addition, the heme pocket of PGHS is much more exposed to the bulk solvent than the heme pocket of MPO. The scan procedure shows that PGHS is designed to accept a large substrate and provides initial guidance for the placement of a large substrate. A substrate fragment containing the five-membered-ring headgroup of PGG2 without the endoperoxide moiety and two short carbon tails emanating from the ring, with one a threecarbon tail having a terminal hydroperoxide group and the other tail a methyl group, was generated in MOE. Preparation for substrate docking consisted of first positioning a docking box that encompassed the test spheres in the peroxidase distal-heme site. Using this docking box as a guide, we positioned the first substrate fragment in an orientation that led to small, positive (∼70 kcal/mol) van der Waals energy between the protein and the substrate fragment. In addition, the magnitudes of the charges on the terminal R oxygen (Figure 4) of the peroxide substrate fragment and the heme iron were increased above that assigned by CHARMm22 to enhance the probability of bringing the peroxide group toward the iron. The placement of this first fragment was then fine tuned by using the simulated annealing software package MOE-DOCK. The docking procedure used several runs where each run consisted of many docking iterations at a temperature of 1 K. The temperature was kept low to reduce the occurrence of multiple conformations with large-scale geometric changes. After the formation of several ligand conformers in a single run, we set aside the fragment with the lowest docking energy in a database. Multiple runs were carried out, leading to many docked conformers. From these, a best-fit ligand-protein conformer was chosen on the basis of the van der Waals contact energy between the substrate fragment and the protein. This best-fit ligand-protein complex of the current series of runs was then selected for the next series of runs. This procedure was iterated until the docking energy (van der Waals energy) stabilized at some negative value of a few kcal/mol. Subsequently, this first substrate-protein construct was modified by

Docking/Molecular Dynamics of a PG Analog

Figure 5. Distances between key hydrophobic residues in the distal peroxidase pocket and the heme iron of PGHS versus time.

the addition of more atoms to the carbon tails (with the hydroperoxide in the S configuration) and redocked using the same protocol outlined above. The procedure was continued until a final PGG2-like substrate, called pseudo-PGG2 (Figure 4), was docked to PGHS. Interestingly, as the size of the ligand was increased, there was a tendency for the docking energy of the best-fit structure to decrease. For pseudo-PGG2, the final energy was ∼ -11 kcal/mol. The docked pseudo-PGG2 has its terminal peroxide oxygen 3.2 Å from the heme iron and >3.5 Å from both the distal His207 NE and Gln203 OE. The carboxylate tail of the pseudoPGG2 extends to Phe409 of the enzyme and into the solventaccessible space. The other carbon tail of pseudo-PGG2, the ω-carbon tail, containing the hydroperoxide oxygens, does not penetrate into the heme pocket past the distal His207 because of a lack of space. Instead, the terminal carbon atoms C16C20 form a U-shaped structure and turn around over the plane of the heme with the tail pointing toward the solvent but not reaching it. Molecular Dynamics of the Pseudo-PGG2 PGHS Complex. The pseudo-PGG2-PGHS complex docked as described above was the starting system for MD. The calculated (see Methods section) atom charges were now added to the appropriate atom types, and the simulation was run for approximately 2 ns. Within the first 60 ps, the docked substrate moved further into the distal heme interior with both of the hydroperoxide oxygens positioned to coordinate with the heme iron, forming the sixth iron ligand, and to hydrogen bond to the distal His207. Notable interactions between pseudo-PGG2 and PGHS that occurred during the simulation are as follows: 1. Hydrophobic Interactions. The pseudo-PGG2-PGHS simulation resulted in significant movements of a few of the distal residues, here Val291, Leu294, and Phe409. The hydrophobic side chains of Val291 and Leu294, which are located in the distal pocket over the heme group, show initial movements (within 60 ps) away from their crystallographic positions. Monitoring the distances of these residues from the heme iron, as displayed in Figure 5, shows that Val291 first moves away from its crystallographic position but then gradually moves to ∼8 Å (measured from the CB side-chain atom) from the iron by the end of the simulation. The distance between the Leu294 side-chain CG atom and the heme iron increases from 10 Å initially to 15 Å and then decreases to 12 Å by the end of the simulation. Conversely, Phe409 moves away from the heme iron throughout the entire MD simulation until the final distance is

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Figure 6. Distances between the heme iron of PGHS and the R (dashed line) and β (solid line) peroxide oxygens of pseudo-PGG2 versus time.

∼12-13 Å between the iron and the CG atom of Phe409. Most of the other heme pocket residues, by comparison, exhibit only small movements during the simulation. The overall secondary/ backbone structure of PGHS shows very little movement away from that of the crystal structure, with the larger movements seen in the region corresponding to the membrane binding domain. When the crystal structure and final MD structure are overlayed, the rms deviation for the main-chain atoms is 1.6 Å. 2. Iron-Oxygen Interactions. The hydroperoxide group of pseudo-PGG2 positions itself more snugly into the POX active site within the first 60 ps of MD (Figure 6). Once the hydroperoxide oxygen atoms assume a catalytic distance (2 to 3 Å) from the iron, they begin exchanging coordination positions throughout the MD. This behavior ensures that at least one oxygen of the hydroperoxide group is 3.1 Å. Interestingly, the distal His207 dihedral angle motions for the water and substrate pseudo-PGG2 simulations differ substantially (Figure 8). The substrate-free simulation has the His207 imidazole ring oriented perpendicular to the heme plane and forming a 75° angle with a plane perpendicular to the heme plane that bisects the propionates. In contrast, the corresponding

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Figure 9. Position of the carboxylate tail (C1-C7) of pseudo-PGG2 after the 2-ns simulation. The carboxylate group always interacts with waters (space filling) of the bulk solvent. Residues labeled by color: Gln203 green, His207 dark blue, Phe409 blue-green, Leu294 purple, Pro276 light blue, and Arg277 red.

Figure 7. Distances between the hydrogen bonding side chain nitrogens of His207 (solid line) and Gln203 (dashed line) and the R (terminal) oxygen of pseudo-PGG2 bound at the peroxidase active site of PGHS versus time.

Figure 10. Contacts between residues in the distal pocket of the POX site of PGHS and C13-C20 of the hydrophobic ω-carbon tail of pseudo-PGG2 after 2 ns of simulation. The closest contacts between C13-C20 (space filling) and the protein interior are made with the side-chain atoms of Lys211 (blue), Gln289 (green), and Val291 (orange). The peroxide group is represented by red spheres.

Figure 8. Orientation of the distal His207 imidazole ring dihedral angle (CA-CB-CG-ND1) with bound water (solid line) or pseudo-PGG2 (dashed line) at the heme active site.

angle for the pseudo-PGG2-PGHS simulation is 25°. Around these origins, the His207 dihedral defining the imidazole plane rocks with about a 50° amplitude for the substrate-free simulation, whereas that of the pseudo-PGG2-PGHS simulation, over the same time period, exhibits only a 30° fluctuation. The substrate simulation that was run longer exhibits the same behavior over the remainder of the trajectory. 4. Pseudo-PGG2. As in the native substrate, pseudo-PGG2 has a five-membered carbon-ring headgroup and two long carbon tails, with one terminating in a methyl group and the other with a carboxylate group. During the MD simulation, the most flexible of these three moieties was the chain containing the carboxylate. This group maps out a large section of the accessible space in its vicinity, as determined by the search algorithm described in the Methods section. The carboxylate tail moves in, relative to the docked structure, to improve the contacts with both Phe409 and Leu408 and makes a 90° angle with the A-ring propionate at the beginning of the simulation. As MD continues, eventually the carboxylate tail migrates from this 90° orientation relative to the A-ring propionate, and adjacent to Phe409, up toward Leu295, where the tail now

Figure 11. Orientation and protein residue contacts of the fivemembered ring (in yellow) of pseudo-PGG2 after 2 ns of simulation. The ring reorients to this position at approximately 500 ps, resulting in more protein contacts, consisting of the six residues Val291 (orange), Leu294 (dark purple), Leu295 (light purple), Leu298 (light blue), Leu408 (green), and Phe409 (blue-green), that persist throughout.

makes a 45° angle with the A-ring propionate and is farther from Phe409 (Figure 9). Throughout the simulation, the carboxylate tail continually makes hydrogen-bonding contact with bulk water. The ω-carbon tail, containing the 15S-hydroperoxide group, (Figure 4) samples much less space and consistently interacts with Val291, Phe292, Leu298, Leu295, and the long carbon side chains of Lys211 and Gln289 (Figure 10). The snapshot in Figure 10 corresponds to the same time as that of Figure 9. After 400 ps, the headgroup flips (rapidly) toward the interior of the heme pocket. As a consequence, distal residues Phe409, Leu295, Leu294, and Leu408 corral the five-membered ring, restricting its mobility for the duration of the simulation (Figure 11). The snapshot in Figure 11 was also taken at the same time as that of Figures 9 and 10.

Docking/Molecular Dynamics of a PG Analog Discussion Substrate-Free Enzyme. The substrate free simulation is in agreement with previous crystallographic,12 spectroscopic,11 and computational studies25 that concluded that a water is the sixth iron ligand of PGHS. Water migration into the heme site (Figure 1) results in the formation of water chains consisting of four to five waters that extend from the sixth coordinated (root) water to the bulk solvent. The root water-iron interaction is very stable and persists throughout the simulation. However, interactions involving the other water molecules are less stable, and these waters readily exchange with the bulk solvent, creating new water chains. The water that hydrogen bonds to the root water has a longer lifetime in the water chain than the other waters found in the chain. If the water chain interacts with a neighboring amino acid or backbone nitrogen or carbonyl, then it too has a longer lifetime in the chain. The distal His207 hydrogen bonded to the root water throughout the simulation and Gln203 had periodic interactions with the root water. In addition, the A-ring propionate makes hydrogen-bonding contact with bulk waters and, at times, participated in the hydrogenbonded water chain. This is probably the scenario for many peroxidases. Crystallographic studies of other peroxidases such as MPO13 and lignin peroxidase (LIP)28 have shown the presence of water in the heme pocket with a water ligating to the heme iron. There are spectroscopic observations of water ligation to the heme iron in other peroxidases.29 Thus, there is the possibility of water ligation to the heme iron and the formation of water chains in these enzymes. However, in the case of MPO and LIP, direct water exchange with bulk water would not be expected to occur because their heme-containing active sites are in the interior of the protein, as opposed to PGHS where the heme has a large area of direct contact with bulk water. One role of water in the active site may be to position various amino acids on the distal side of the heme for the incoming substrate. Our simulation shows that the incorporation of water into the active site is followed by an expansion of the heme pocket, as seen by the movement of certain hydrophobic side chains lining the active site (data not shown). Such an expansion of the distal pocket is most likely required for docking the substrate into a conformationally functional site. However, it has also been postulated that the water in the active site might interfere with an incoming substrate such as hydrogen peroxide.30 In the case of LIP, this effect may hinder both the access of the hydrogen peroxide substrate and the enzyme reducing agents at the heme active site and influence the production of compound I.31 Pseudo-PGG2 Docking Simulations. To investigate the ability of PGHS to accommodate a large substrate such as PGG2, we carried out docking studies were with the goal of first docking a substrate fragment and then, once docked, building onto this fragment with repeated docking at every stage until a realistic substrate very similar to PGG2 would be obtained. The first attempts at docking substrate fragments consisting of the headgroup only or a carbon chain containing a peroxide were unsuccessful because of escalating van der Waals contact energies and catalytically unproductive conformations. This motivated the construction of the sphere search algorithm (see Methods section) that locates van der Waals space for ligand placement within the protein. The result showed that PGHS has an open heme pocket with good accessibility to the bulk solvent (Figure 2a). There is also a space that extends from the heme plane to Phe409, approximately 12 Å away, which is also accessible to the test spheres. However, we found that no available space was present for docking from the distal His207

J. Phys. Chem. B, Vol. 108, No. 26, 2004 9303 to the edge of the heme facing the protein interior (on the opposite side of the heme from the propionates). Thus, about one-half of the distal heme surface is available for substrate docking. In contrast, MPO, a structurally related peroxidase, has much less space available on the distal side of the heme pocket and, hence, could not embed a large substrate (Figure 2b). This is consistent with the preferred substrate of MPO being hydrogen peroxide,14 a much smaller substrate than PGG2. Interaction of Pseudo-PGG2 with the Heme Pocket of PGHS. The movement of distal residues Val291, Leu294, and Phe409 from their original (crystallographic) positions during the initial MD simulations illustrates that the heme pocket expands to accommodate PGG2 (Figure 5). This allows pseudoPGG2 to move into the active site where it increases its number of hydrophobic contacts. Once the pseudo-PGG2 substrate enters the heme pocket, both Val291 and Leu294 move closer to the substrate, positioning themselves to within 8.5 and 12 Å, respectively. The insertion and interaction of the substrate with PGHS most likely excludes not only the ligated water but also all waters because pseudo-PGG2 occupies a significant portion of the volume of the heme pocket. In addition, during the entire simulation, water molecules move around the heme site, permeating some interior sites but never penetrating the heme pocket. 1. Interactions InVolVing the FiVe-Membered Ring of PseudoPGG2. After ∼400 ps, there is a flip of the five-memberedring headgroup of pseudo-PGG2, resulting in a change from tilting toward the bulk solvent to facing the protein interior (Figure 11). The movement acts like a packing process where the five-membered ring now faces the interior hydrophobic pocket, where it is corralled by Val291, Leu294, Leu298, Leu295, Leu408, and Phe409. In this configuration, pseudoPGG2 reduces its contact with the bulk water and increases its interaction with the heme pocket, probably leading to a more tightly bound structure. The reorientation of the headgroup had no effect on the hydrogen bonding of His207 and the ironperoxy interaction. It is important to note, in examining the structures in Figures 9-11, that pseudo-PGG2 is oriented in the peroxidase site such that the endoperoxide group, were it attached to pseudo-PGG2, would face away from the protein. We suggest that the carbons of the five-membered ring would be expected to interact with the protein in much the same way as with pseudo-PGG2 or with naturally occurring PGG2. 2. Interactions InVolVing the Fatty Acid Chains of PseudoPGG2. The side chain containing the carboxylate group of pseudo-PGG2 moves throughout the simulation, sampling a great deal of the available space mapped by the test spheres. The carboxylate tail moves from its interior position where it makes contact with Val291, Phe409, Leu408, and Leu295 and the proximal residues Ile444 and Val447 to one in which it replaces the contact Leu295 and the proximal residues with contacts involving Leu294, Pro276, His274, and Tyr275 (Figure 9). These latter three residues reside at the lip of the POX pocket where they are exposed to the solvent. Thus, PGHS allows for flexibility in the carboxylate tail of pseudo-PGG2 so that it can associate and move with the bulk water. The carbon chain containing the 15S-hydroperoxide group moves to a much lesser extent than the carboxylate tail, maintaining its van der Waals contacts with the side chains of Lys211, Gln289, Val291, Leu295, and Phe292 throughout the simulation (Figure 10). The NZ atom of Lys211 and the oxygens of Gln289 are in hydrogen bonding contact with each other and form a barrier, separating water on one side and interacting with the peroxy hydrocarbon tail of pseudo-PGG2 on the other,

9304 J. Phys. Chem. B, Vol. 108, No. 26, 2004 utilizing their polar headgroups and hydrocarbon side chains, respectively. The hydrophobic side chains of Val291, Leu295, and Phe292, located over the heme plane, make van der Waals contacts with the ω-carbon tail, restricting its ability to move further into the heme pocket. Thus, the ω-carbon tail is much less mobile than the carboxylate tail. Interestingly, pseudo-PGG2 is oriented such that a trans double bond between C13-C14, as occurs in PGG2, would not affect the position of the ring or hydroperoxide. Pseudo-PGG2 and Its Catalytic Configuration. As we pointed out in the Results section, the simulated annealing procedure produced a best-fit pseudo-PGG2-enzyme complex with low van der Waals contact energies, suggesting a feasible substrate orientation with respect to PGHS. However, the construct is not a catalytically active species because of the lack of contact between the hydroperoxide group of the substrate with His207 and Gln203 and the heme iron in the distal pocket. This result represents a limitation in the docking of ligands to protein crystal structures and illustrates the necessary involvement of protein motion in substrate binding. Once the distal amino acids allow substrate penetration into the pocket during the simulation, the peroxide makes hydrogen-bonding contact with the distal His207 (Figure 7) and ligates at the sixth coordination position of the iron (Figure 6). The hydrogen bonding here is very strong, as indicated by the reduced angular movement of the distal histidine when compared to the analogous movement in the substrate-free simulation (Figure 8). The R and β oxygens rock back and forth, exchanging ligation with the iron (Figure 6), a behavior that has been noted in previous studies.25,30 The combination of the position of the hydroperoxyl group of pseudo-PGG2 and its distal interactions produces a catalytically active conformation that is consistent with the geometric requirements of the push-pull mechanism.9,10 In this mechanism, it is essential that the peroxide R-oxygen hydrogen bond to the distal histidine to perform the acid-base chemistry necessary for the pull involved in the cleavage of the oxygen-oxygen bond. This distal His207 is required for peroxidase catalysis by both PGHS-132 and PGHS2.33 The distal Gln203 NE nitrogen moves very early in the simulation to within hydrogen-bonding distance of the R oxygen of the hydroperoxyl group of pseudo-PGG2. This result is consistent with the idea that Gln203 participates in hydrogen bonding with the ferryl-oxo species to stabilize compound I and intermediate II.34 However, in the function of PGHS-1, the role of Gln203 in peroxidase catalysis has not been examined directly. In PGHS-2, asparagine but not arginine or valine can substitute for Gln203.33 Thus, the precise role of Gln203 in catalysis has not been established. In conclusion, our goal in this study was to find a configuration of pseudo-PGG2 that could be viewed as catalytically competent on the basis of the push-pull mechanism of peroxidase activity, which requires the peroxide oxygens of the substrate to be within ligation distance of the heme iron and for the R-peroxide oxygen to be able to hydrogen bond to the distal residues His207 and Gln203. A combination of the docking protocol we developed and MD simulation led to a catalytically competent substrate configuration, with the mobile, carboxylate-terminated tail oriented toward the bulk water. Having the carboxylate tail within easy access of the bulk solvent could facilitate substrate entry into and out of the binding site. The hydroperoxide tail of pseudo-PGG2 is much less mobile and becomes, as the simulation proceeds, surrounded by a number of hydrophobic residues as well as by the nonpolar side-

Seibold et al. chain atoms of Lys211. The headgroup also becomes protected from the solvent by a number of hydrophobic residues. Thus, the peroxidase site of PGHS seems well designed to accommodate a large hydroperoxide substrate. Acknowledgment. This work was supported in part by NIH grants P01 GM57323, R01 GM68848, and R01 GM 47274. Abbreviations PGHS, prostaglandin endoperoxide H synthase; MD, molecular dynamics; MPO, myeloperoxidase; PGG2 15-hydroperoxy9,11-endoperoxide prostaglandin G2; COX, cyclooxygenase site; POX, peroxidase site; LIP, lignin peroxidase; PGH2, alcohol product of the hydroperoxide PGG2; AA, arachidonic acid. References and Notes (1) Smith, W. L.; Garavito, R. M.; DeWitt, D. L. Annu. ReV. Biochem. 2000, 69, 145. (2) Smith, W. L.; Song, I. Prostaglandins Other Lipid Mediators 2002, 68-69, 115. (3) Rouzer, C. A.; Marnett, L. J. Chem. ReV. 2003, 103, 2239. (4) Marnett, L. J.; Rowlinson, S. W.; Goodwin, D. C.; Kalgutkar, A. S.; Lanzo, C. A. J. Biol. Chem. 1999, 274, 22903. (5) Lambeir, A. M.; Markey, C. M.; Dunford, H. B.; Marnett, L. J. J. Biol. Chem. 1985, 260, 4894. (6) Karthein, R.; Dietz, R.; Nastainczyk, W.; Ruf, H. H. Eur. J. Biochem. 1988, 171, 313. (7) Koshkin, V.; Dunford, H. B. Biochim. Biophys. Acta 1999, 1431, 47. (8) van der Donk, W. A.; Tsai, A. L.; Kulmacz, R. J. Biochemistry 2002, 41, 15451. (9) Poulos, T. L. J. Biol. Inorg. Chem. 1996, 1, 356. (10) Dawson, J. H. Science 1988, 240, 433. (11) Seibold, S. A.; Cerda, J. F.; Mulichak, A. M.; Song, I.; Garavito, R. M.; Arakawa, T.; Smith, W. L.; Babcock, G. T. Biochemistry 2000, 39, 6616. (12) Picot, D.; Loll, P. J.; Garavito, R. M. Nature 1994, 367, 243. (13) Fiedler, T. J.; Davey, C. A.; Fenna, R. E. J. Biol. Chem. 2000, 275, 11964. (14) Furtmuller, P. G.; Burner, U.; Jantschko, W.; Regelsberger, G.; Obinger, C. FEBS Lett. 2000, 484, 139. (15) Ohki, S.; Ogino, N.; Yamamoto, S.; Hayaishi, O. J. Biol. Chem. 1979, 254, 829. (16) MOE. Molecular Operating Environment; Chemical Computing Company: Montreal, Quebec, Canada, ×2003 (17) MacKerell, A. D.; Brooks, B. R.; Brooks, C. L., III.; Nilsson, L.; Rous, B.; Won, Y.; Karplus, M. CHARMM: The Energy Function and Its Parametrization with an Overview of the Program. In the Encyclopedia of Computational Chemistry; von Rague´ Schleyer, P., Ed.; John Wiley & Sons: Chichester, U.K., 1998; Vol. 1, p 271. (18) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.11.1; Gaussian, Inc.: Pittsburgh, PA, 1998. (19) Schafmeister, C. E. A. F.; Ross, W. S.; Romanovski, V. LEaP Software Package; University of California: San Francisco, CA, 1995. (20) Loll, P. J.; Sharkey, C. T.; O’Connor, S. J.; Dooley, C. M.; O’Brien, E.; Edvocelle, M.; Nolan, K. B.; Selinsky, B. S.; Fitzgerald, D. J. Mol. Pharmacol. 2001, 60, 1407. (21) Berendsen, H. H. C.; Postma, J. P. M.; Gunsteren, W. F.; DiNola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684. (22) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, G. L. J. Chem. Phys. 1995, 103, 8577. (23) Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham, T. E., III; Wang, J.; Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.; Stanton, R. V.; Cheng, A. L.; Vincent, J. J.; Crowley, M.; Tsue, V.; Gohlke,

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