The structures of prostaglandin endoperoxide H synthases-1 and -2

Prostaglandins & other Lipid Mediators 68–69 (2002) 129–152

The structures of prostaglandin endoperoxide H synthases-1 and -2 R. Michael Garavito a,∗ , Michael G. Malkowski b , David L. DeWitt a a

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA b Department of Biochemistry and Molecular Biology, University of Buffalo, Buffalo, NY, USA

Abstract Despite the marked differences in their physiological roles, the structures and catalytic functions of the prostaglandin H2 endoperoxide synthases-1 and -2 (PGHS-1 and -2) are almost completely identical. These integral membrane proteins catalyze the conversion of arachidonic acid to PGG2 and finally to PGH2 . The crystal structures of PGHS-1 and -2 provide new insights into the catalytic mechanism for fatty acid oxygenation. Moreover, a clearer picture emerges to explain how a handful of amino acid substitutions can give rise to subtle differences in ligand binding between the two isoforms. These “small” alterations of isozyme structure are sufficient to allow the design of new, isoform-selective drugs. © 2002 Elsevier Science Inc. All rights reserved. Keywords: X-ray structure; Cyclooxygenase; Peroxidase; Fatty acid binding; Arachidonic acid; Aspirin; Nonsteroidal antiinflammatory drugs; COX-2 selective inhibitors; Drug binding

1. Introduction The enzyme prostaglandin endoperoxide H2 synthase (PGHS; EC 1.14.99.1) is a membrane bound, heme-dependent bis-oxygenase (cyclooxygenase) and hydroperoxidase. Also known as prostaglandin H2 synthase or cyclooxygenase (COX), PGHS catalyzes the committed step in prostanoid synthesis [1–4] via two sequential enzymatic reactions: (1) the bis-oxygenation of arachidonic acid (COX) forms PGG2 and (2) reduction of 15-hydroperoxide of PGG2 in the peroxidase (POX) active site to form PGH2 . Prostanoids are members of a large group of bioactive, oxygenated C18 –C22 compounds that are derived from ␻3 (n−3) and ␻6 (n−6) polyunsaturated fatty acids. Prostanoids are a subclass of compounds collectively known as eicosanoids [5] and are formed through cyclooxygenase pathway. In mammals, arachidonic acid (20:4n−6) is the major prostanoid precursor. ∗

Corresponding author. Tel.: +1-517-355-9724; fax: +1-517-353-9334. E-mail address: [email protected] (R. Michael Garavito).

0090-6980/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 0 2 ) 0 0 0 2 6 - 6

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Two isoforms of PGHS are found in mammals [1,6]: the “constitutively” expressed PGHS-1 and the “inducible” PGHS-2. Both enzymes are present on the lumenal surfaces of the ER and of the inner and outer membranes of the nuclear envelope [7,8]. Understanding the structure and function of the PGHS isoforms has been the focus of much recent research because both isoforms are the targets of nonsteroidal antiinflammatory drugs (NSAIDs). After the discovery of the PGHS-1 and -2, it quickly became apparent that the isoforms were noticeably different in their expression profiles and roles in several physiological processes [1,6]. The PGHS isozymes also have roles in a wide range of pathologies that include, for PGHS-1, thrombosis [9,10] and for PGHS-2, inflammation, pain and fever [11], various cancers [12,13] and neurological disorders like Alzheimer’s [14] and Parkinson’s [15] diseases. Within the last decade, pharmacological research has led to the development and approval of the new “COX-2” selective inhibitors Celebrex® and Vioxx® which target PGHS-2. In this chapter, we will discuss the structures of the PGHS isoforms and their relevance to the pharmacology and enzymology of these enzymes. The reader is directed to the other chapters in this issue that discuss, in more detail, the molecular biology, biochemistry, pharmacology and enzymology of the PGHS isoforms.

2. Primary structure of prostaglandin endoperoxide H2 synthase (PGHS) The primary structures of PGHS-1 and -2 from several mammalian species and chicken [16] show that nascent PGHS-1 and -2 are processed into mature enzyme forms containing 576 amino acids and 587 amino acids, respectively. The numbering of amino acids begins at the N-terminal methionine of the signal sequence which is removed in the mature protein [2]. The high degree of sequence identity between the mature isoforms and between species allows an almost one-to-one comparison between all known sequences, aside from small insertions and deletions in PGHS-2s [1,2]. To aid in cross-isoform and cross-species comparisons, we will use the ovine PGHS-1 sequence numbering as the reference, which results in renumbering of the PGHS-2 sequences by adding 15 initially. Hence, the N-terminal residues of PGHS-1 and PGHS-2 are then numbered Ala25 and Ala33, respectively (Fig. 1), after cleavage of the signal sequence. Sequential numbering then continues until the first and only insertion within the main portion of the protein sequence: a proline residue found in PGHS-2 after Thr106 has no counterpart in PGHS-1 and is thus referred to as Pro106A. For the rest of the primary sequence, the numbering is sequential for both isoforms until the C-terminus, where an 18-amino acid insertion occurs in PGHS-2 [16]. Thus, the Arg106 of PGHS-2 is numbered as Arg120 as in PGHS-1 for convenience of structural and functional comparisons. PGHS-1 and -2 from the same species display 60–65% sequence identity while sequence identity among orthologs from different species varies from 85–90% [16]. The major differences in primary structure between PGHS isoforms occur in four distinct areas of the sequence (Fig. 1). First, both isoforms contain signal peptides of varying lengths. Second, an 18-amino acid insertion occurs six residues in from the C-terminus of PGHS-2 that are not present in PGHS-1. The function of this insertion in PGHS-2 is not known, but it may possibly be a “signal domain” for controlling protein turnover or subcellular trafficking. Elimination of this cassette by deletion mutagenesis has no apparent effect on PGHS-2 catalysis (DeWitt and Smith, unpublished observations). Third, substantial sequence

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Fig. 1. A schematic diagram of the PGHS-1 and -2 sequences with structural landmarks denoted; upon renumbering, the landmarks are numbered identically. Both isoforms have an epidermal growth factor (EGF)-like domain, a membrane-binding domain (MBD), and a catalytic domain. PTEL in PGHS-1 and STEL in PGHS-2 refer to the final four residues in each sequence and suggest an ER retention signal [81].

differences are found in the membrane-binding domains (MBDs) between the two isoforms [8,17], although no explanation for this phenomenon is known. Finally, PGHS-1 is N-glycosylated at three sites, while PGHS-2 is variably glycosylated at two to four sites [18]. N-glycosylation of PGHS-1 seems to be required for enzyme folding [18], and the need for proper N-glycosylation of PGHS-1 created problems in producing large quantities of this isoform by recombinant methods [19]. This contrasts with successes with expressing PGHS-2 in baculoviral-insect cell systems [19–21]. A consequence of the heterogeneous glycosylation in PGHS-2 is that multiple molecular species can be readily observed with SDS-PAGE.

3. Characteristics of purified PGHS Both PGHS isoforms are integral membrane proteins and all large-scale isolation protocols using tissue [22–24]or insect cell expression of recombinant protein [19–21] rely on the preparation of microsomes from freshly homogenized material, whereupon subsequent detergent solubilization releases the enzyme from the bilayer [19,23]. Detergent-solubilized PGHS-1 and -2 appear as homodimers in solution and as 67–72 kDa monomers on SDSPAGE [1,2], depending on isoform and heterogeneity of glycosylation. Both PGHS isoforms bind 1 mol of ferric-protoporphyrin IX per mole monomer for full activity. A judicious choice of detergent for solubilization and purification of ovine PGHS-1 can result in retention of the bound heme or its loss [22,23]. For recombinant PGHS-2 expressed in insect cell culture [19–21], detergent solubilization and subsequent purification tends to yield apo-enzyme, suggesting that the heme affinity is lower for this isoform than for PGHS-1. Reconstitution of the active holo-enzyme, for either PGHS-1 or -2, is easily done with the addition of hematin to the apo-enzyme. Moreover, the heme in PGHSs can

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be readily replaced by mangano-protoporphyrin IX or cobalt-protoporphyrin IX to create holo-species that are structurally native, but have quite altered activity [23,25–27]. N-terminal His-tagged versions of human PGHS-1 and -2 have been prepared and expressed in high yield in insect cell culture [19]. The same methodology has now been applied to ovine PGHS-1 as well (Malkowski, Smith, DeWitt, and Garavito, unpublished results). In all cases, the addition of the N-terminal hexaHis-tag, between the signal peptide and the growth factor-like (EGF) domain, does not apparently impact the folding of the heterologously expressed enzyme or its activity. This methodological development now allows for facile and rapid purification of active native and mutant PGHS enzyme for enzymological and structural research.

4. The crystal structure of PGHS The three dimensional structure of the ovine PGHS-1 was first reported in 1994 [28] (Fig. 2) and the crystal structures of human [29] and murine [30] PGHS-2 quickly followed. More recently, the several structures of ovine PGHS-1 have been determined while complexed with several different fatty acid substrates: arachidonic acid (AA; 20:4n−6) [31], dihomo-␥-linolenic (DHLA; 20:3n−6) [32], linoleic acid (LA; 18:2n−6) [33], and eicosapentaenoic acid (EPA; 20:5n−3) [33]. Crystal structures of murine PGHS-2 complexed with AA have also been determined [34]. PGHS functions as a homodimer [35] and attempts to create monomeric species have yielded only inactive enzyme (Harlan and Garavito, unpublished results). The successful determination of the PGHS structures depended on obtaining crystals of these integral membrane proteins from detergent-containing solutions. As a consequence, the formation of inactive monomer often occurs due to detergent-induced inactivation and denaturation, phenomena that also adversely affect PGHS crystallization. The crystal structures of the PGHS isoforms are quite structurally homologous and superimposable [29,30], as might be expected from the observed levels of sequence identity. The PGHS monomer (Fig. 3) consists of three structural domains: an N-terminal epidermal EGF domain, an MBD of about 48 amino acids in length, and a large C-terminal globular catalytic domain with the heme binding site facing the solvent. The structures of the C-terminal segments beyond Pro583 (17 amino acids in PGHS-1 and 35 amino acids in PGHS-2) have not been resolved crystallographically [28–30]. Nonetheless, this region of the enzyme is of great interest as PGHS-1 and -2 contain C-terminal KDEL-like sequences that may target PGHSs to the endoplasmic reticulum and the associated nuclear envelope [1]. 4.1. EGF domain The dimer interface is created by the EGF and catalytic domains (Fig. 4), which places the two MBDs in a homodimer about 25 Å apart. The EGF domains clearly make up a substantial and intimate portion of the dimer interface of PGHS. Whether or not the EGF domains have some functional significance is unclear. Certainly, EGF domains are common in several families of membrane proteins and secreted proteins [36]. Typically, the EGF domain occurs at a position in the primary sequence N-terminal to a membrane anchor, such that these domains always occur on the extracytoplasmic face of the membrane.

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Fig. 3. Ribbon drawing of the ovine PGHS-1 monomer with bound arachidonic acid (AA). The locations of the POX active site and the sites of N-linked glycosylation at asparagines N68, N144, and N410. The color scheme for the polypeptide chain is the same as for Fig. 2. Note the AA (yellow) buried within the COX active site and the four molecules of the detergent ␤-octyl-d-glucopyranoside (␤-OG) at the mouth.

Garavito and co-workers [37,38] have suggested that the EGF domains play a role in the integration of maturing PGHS into the lipid bilayer. Consistent with this hypothesis, Li et al. [39] showed that green fluorescent protein can be anchored to bilayers by fusing both the EGF domain and MBD to its N-terminus. Interestingly, mammalian thyroid POX, which is related to PGHS, also uses an EGF domain and a membrane anchor to integrate the POX catalytic domain onto the membrane [40]. 4.2. MBD The MBDs of PGHS isoforms contain four short, consecutive, amphipathic ␣-helices (Fig. 3). Three of the four helices lie in roughly the same plane while last helix merges “upwards” into the catalytic domain. Hydrophobic and aromatic residues protrude from these helices to create a hydrophobic surface which would interact with the one face of the lipid bilayer [28]. The MBDs of PGHS-1 and -2 thus represent the first example of the

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Fig. 4. A space-filling model of the PGHS-1 dimer viewed from the membrane plane. The EGF and MBD domains are colored green and gold, respectively. The catalytic domains are colored two different shades of blue to highlight the dimer interface. Note that how the MBD spirals around the COX channel to make the mouth of the COX active site. Arg120 (purple), which is part of the channel aperture, defines the beginning of the COX active site. Within one COX channel, a buried AA (yellow and red) is shown.

“monotopic” mechanism for integrating into biological membranes. The general functionality of membrane anchoring via monotopic interactions has been studied biochemically [39] and by computer modeling [41]. As mentioned in the previous section, The PGHS isozymes can only be purified to homogeneity using nonionic detergents [20,21,24] and are thus crystallized in their presence. Examples of some tightly bound detergent molecules in the PGHS structures can be easily resolved crystallographically [29,31,42,43] (Fig. 3). 4.3. The catalytic domain The catalytic domain comprises the bulk of the PGHS isoforms [37]. The globular catalytic domain, which is almost entirely comprised of ␣-helical secondary structure, shares a great deal of structural homology with mammalian myeloperoxidase [28,37], a soluble

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heme-dependent POX. Structural homology between the PGHS catalytic domain and nonmammalian heme-dependent POXs is also detectable [28,37,44]. The POX active site in PGHS resides in a large groove on the side opposite of the MBD (Figs. 3 and 5a). The entrance to the COX active site is between the helices of the MBD: this opening leads to a 25 Å long, hydrophobic channel within the interior of the catalytic domain [28] (Figs. 3 and 4). The COX channel contains several side pockets and cul-de-sacs [28–30] as well as a branched water channel [45] which extends from the COX active site near Gly533 to the dimer interface. All fatty acid substrates and NSAIDs must enter the COX active site through the MBD. The very narrow dimensions of the aperture within the COX channel (Fig. 4) clearly suggest that the MBD may undergo significant conformational changes during substrate entry and product exit [43]. Such interior cavities are unknown in other heme-dependent POXs, like myeloperoxidase. From an evolutionary prospective, an ancestral POX must have undergone two distinct changes to create PGHS: (1) the formation of an interior channel for the COX reaction and (2) the acquisition of the membrane binding.

5. The POX active site The POX active sites in the PGHS isozymes are quite open to the solvent in contrast to virtually all other POXs (Fig. 5). In the refined crystal structure of ovine PGHS-1 [45], the POX active site (Fig. 5b) reveals that His388 is the proximal heme ligand: the Nε nitrogen bonds to the ferric iron while the N␦ participates in a hydrogen-bond network involving a water molecule and Tyr504. In PGHS-2, the identical arrangement is seen but the existence of proximal water molecule has not been commented on [29,30]. Thus, the interactions on the proximal heme face in PGHS contrast with other POXs where the proximal histidine forms an ionic bond or strong hydrogen bond with aspartate or asparagine [44,46], respectively. On the distal heme surface, His207 is predicted to be important in the deprotonation of the peroxide substrate and subsequent reprotonation of the incipient alkyloxide ion to form the alcohol during generation of compound I [47]. Gln203 is also important in catalysis, although its function has not been fully resolved [47]. Mutations of Gln203, His207, or His388 in ovine PGHS-1 and human PGHS-2 lead to a reduction or elimination of POX activity [47,48]. Typical heme iron ligands like CO or CN− bind to the distal side of the iron with a linear or “unbent” geometry in ovine PGHS-1 [45], while in myeloperoxidase, the Fe–C–O bond shows considerable bending [49]. While this seems to be a reasonable result as myeloperoxidase has a quite buried active site [50], paradoxically myeloperoxidase readily binds the larger thiocyanate [44] unlike ovine PGHS-1. Thus, how PGHS binds tightly large ligands like 15-HPETE or PGG2 , while exhibiting much reduced affinity towards small ligands like azide, thiocyanate, and H2 O2 , is an unresolved question. The low affinity of N3 − and thiocyanate for PGHSs probably arises from unfavorable interactions with distal “roof” residues in the POX active site (Fig. 5b), but the crystal structures provide little insight into how this occurs. Interactions on the proximal side of the heme are considered to affect the reactivity of the heme iron. The proximal histidine is bonded directly to an asparagine in myeloperoxidase [50] and an aspartate in cytochrome c POX [44], creating a more basic proximal ligand. In ovine PGHS-1, rRaman spectroscopy indicates that His388, the proximal ligand, is clearly

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more neutral in character than corresponding histidines in other POXs. His388 in ovine PGHS-1 participates in a water-mediated hydrogen bond with Tyr504, but mutagenesis of Tyr504 to an alanine results in only a marginal loss in POX activity [45]. Thus, the proximal ligand in PGHS requires no backside hydrogen bond to catalyze peroxidation. However, this difference in acidity of the proximal histidine in native PGHS is clearly not reflected in the rate of POX turnover, which is comparable to those of other heme POXs [51]. The heme binds reversibly to ovine PGHS-1 with a Kd ∼1 ␮M. The Kd for heme binding to PGHS-2 is estimated to be even higher based on the relative ease with which apo-PGHS-2 is formed [19–21]. This has allowed the relatively facile removal of heme to create apo-enzyme and the reconstitution of pseudo-holoforms of PGHS with protoporphyrin IX containing different metals. Although Fe3+ -protoporphyrin IX is the natural heme ligand, Mn3+ -protoporphyrin IX can slowly undergo the changes in redox state needed for POX catalysis and can initiate the COX reaction [26,27]. Other metals (e.g. Zn or Co) in protophorphyrin IX do not support either the POX or COX reactions [25]. However, ovine PGHS-1 reconstituted with Co3+ -protoporphyrin IX does form a native-like, albeit inactive, enzyme form with the metal bound in a six-coordination state [23]. This form of the enzyme has been useful for creating stable complexes of ovine PGHS-1 with fatty acid substrates for crystallographic analysis [23,31–33].

6. The COX active site The hydrophobic channel that extends from the MBD to the core of the globular domain can be divided into two distinct regions: the channel “mouth” and the COX active site [43]. The rim and inner surface of the MBD forms the mouth of the channel through which arachidonate and O2 enter directly from the lipid bilayer. The outer rim of the COX channel mouth is a relatively open structure (Fig. 3) in all PGHS crystal structures [52]. The COX catalytic center encompasses the upper half of this channel, extending from Arg120 to Tyr385 (Figs. 3 and 6). At the level of Arg120 and Tyr355, the channel narrows to form a constriction or “aperture” [42,43,52]. All the crystal structures of PGHS-1 and -2 reveal this aperture to be in a seemingly “closed” conformation [52]. The narrowness of this constriction clearly suggests that a major conformational change must occur to allow ingress and egress of ligands into and from the COX active center (see the last section). 6.1. Arachidonic acid bound in the COX site AA is oriented within the COX active site in an extended “L-shaped” conformation (Fig. 6). The apical portion of the substrate binds such that the carboxylate interacts with the guanidinium group of Arg120, a known determinant of substrate binding to PGHS-1 [31,54,53]. Carbons 7–14 of AA form an “S shape” that weaves the substrate chain around the side chain of Ser530, the residue acetylated by aspirin [42,55]. In this conformation, AA is positioned such that carbon 13 is oriented near the phenolic oxygen of Tyr385 (Fig. 6), where the proS hydrogen can be abstracted to initiate the COX reaction. Moreover, carbon 11 is positioned above a small pocket, surrounded by Val349, Ala527, Ser530, and Leu531, into which O2 could migrate from the lipid bilayer [31]. The addition of oxygen to the developing

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Fig. 6. A stereo diagram of the kinked L-conformation of AA (yellow) bound in the COX active site of ovine PGHS-1 [31]. Note that how Tyr385 (copper) is aligned with carbon 13 in AA, and how the carboxylate of arachidonate interacts with the guanido group of Arg120 (see text). Several other residues discussed in the text are also shown; the asterisk (∗ ) refers to the position of Gly533.

arachidonyl radical would thus come from the side opposite of hydrogen abstraction, which is also a known aspect of the COX reaction mechanism [1,3,56]. The ␻-end of AA (carbons 14–20) binds in a hydrophobic groove above Ser530, where it is stabilized by Phe205, Phe209, Val344, Phe381, and Leu534. AA makes a total of 48 van der Waals contacts and two hydrophilic contacts with 19 residues that line the cyclooxygenase active site channel [31]. While PGHS-1 synthesizes primarily PGG2 , it also produces small but significant amounts of 11R-hydroperoxy-(5Z,8Z,12E,13Z)-eicosatetraenoic acid (11R-HPETE) and 15-hydroperoxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15R-HPETE and 15S-HPETE) as inferred from the appearance of the POX-derived hydroxy products, 11R-HETE, 15R-HETE, and 15S-HETE, in product assays [57–59]. The kinetics of the product formation suggests that AA may adopt at least four slightly different but catalytically competent conformers in the COX active site [59]. Human PGHS-2 was also thought to form only PGG2 , 11R-HPETE, and 15S-HPETE but not 15R-HPETE [60], which suggested that there are only three catalytically competent conformers of AA in this isoform. However, more recent work by Schneider et al.[57] shows that wild-type PGHS-2 does form 15R-HPETE. Hence, the structure of AA bound in the COX active site [31] may be a time- and space-average of two or more AA conformers, of which only the predominant conformer leads to PGG2 formation. 6.2. Alternative fatty acid substrates bound in the COX site DHLA (20:3n−6), LA (18:2n−6), and EPA (20:5n−3) also bind to the COX active site in extended L-shaped conformations that are generally similar to that observed for AA (Fig. 7) [32,33]. These alternative substrates have their carboxylate groups positioned such that they make the critical salt bridge with Arg120, and their ␻-ends are placed in

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Fig. 7. Comparison of the binding conformations in ovine PGHS-1 for the fatty acids DHLA (20:3n−6), LA (18:2n−6), and EPA (20:5n−3) in red, green, and light blue, respectively. Also AA is also shown in yellow. The different modes of binding are viewed with respect to the critical residues Tyr385, Arg120, Val349, and Ser530. Note that how the carboxylate ends of the fatty acids exhibit markedly different conformations, while the ␻-ends adopt almost the equivalent conformations (a). The positioning of carbon 13 (carbon 11 for LA) is viewed with respect to Tyr385. While the 13proS hydrogen of AA is well positioned for abstraction by the radical on Tyr385, misalignment of the respective carbons in DHLA, LA, and EPA displaces the appropriate hydrogen farther away from Tyr385. Thus, hydrogen abstraction is more difficult for these fatty acids (b).

the hydrophobic groove above Ser530. Additionally, the central carbons of each substrate are woven around Ser530 resulting in the positioning of carbon 13 (or carbon 11 of LA) below Tyr385 for hydrogen abstraction (Fig. 7b). DHLA, EPA, and LA make 62, 61, and 47 contacts, respectively, with 20 residues lining the COX site [32,33]. The contacts and residues involved in stabilization of the fatty acids within the active site are virtually identical to those observed for AA. Despite a grossly similar binding conformation, the chemical differences in polyunsaturation and carbon length presented by DHLA, EPA, and LA lead to differences in the local structure of each fatty acid compared to AA (Fig. 7a). DHLA lacks the carbon 5/carbon 6 double bond present in AA, which leads to a substrate with greater conformational flexibility in the carboxyl half of the molecule. Specifically, the positions of carbons 2–10 in DHLA differ by up to 1.7 Å versus AA, whereas carbon 1 and carbons 11–20 are in the same positions for both substrates [32]. As a result, the carboxyl half of DHLA takes on a more compact, coiled structure that allows for the required Arg120–carboxylate interaction and the positioning of carbon 13 below Tyr385 for hydrogen abstraction. In contrast, the additional double bond at the ␻-end of EPA makes it a more rigid fatty acid substrate. EPA, thus binds in the cyclooxygenase active site in a “strained” orientation.

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Carbon 1 is oriented to make the carboxylate interaction with Arg120, but the remaining carbons form a more compact conformer and are displaced from their positions assumed in AA. The displacement of these carbons is needed to accommodate the conformationally inflexible ␻-end in the hydrophobic groove above Ser530 [33]. As a consequence, carbon 13 of EPA is significantly misaligned with respect to Tyr385 (Fig. 7b). LA (18:2n−6) is two carbons shorter and contains two fewer double bonds than AA (20:4n−6). Carbons 1–6 of LA are positioned similarly to the equivalent carbons in AA, which maintains the Arg120–carboxylate interaction. However, carbons 7–9 are in a more conformationally extended state, which positions carbon 11 (instead of carbon 13) below Tyr385 at the top of the active site [33]. Other fatty acids like 18:3n−6 and 20:2n−6 should bind in a similar manner which would permit removal of the n−8 hydrogen; 18:3n−3, however, would need to be aligned for abstraction of the n−5 hydrogen [61,62].

7. The COX active site and mutagenesis The structures of PGHS complexed with fatty acids [31–34] have provided a physical backdrop for the interpretation of mutagenic studies on active site residues that investigate the structural steps leading to PGG2 formation. Crystallographic evidence strongly suggests equivalent roles for homologous residues in the PGHS isoforms. However, mutagenic studies clearly reveal that the isoforms behave in distinctly different ways. While the hydrophobic residues in the PGHS-1 COX active site function to position AA in the productive conformer, Arg120 is the critical contributor to substrate binding [53,54,63,64]. The substitution of Arg120 [53,63] markedly alters substrate affinity: the R120Q mutant of oPGHS-1 show a 500–1000-fold higher Km for arachidonate than that measured for native ovine PGHS-1 [53]. In sharp contrast, the R120Q mutation in human PGHS-2 has little effect on Km or Vmax [54]. These results suggest that the hydrophobic residues in the PGHS-2 COX channel must play a more significant role in substrate binding than in PGHS-1. How they compensate for the surprisingly diminished role of Arg120 in PGHS-2 remains unclear. Tyr355 lies on the opposite side of the channel from Arg120 (Fig. 6) and clearly ligands to NSAIDs and substrate in the crystal structures [28–31,42,43]. In contrast to Arg120, mutating this residue has only a very modest effect on substrate–enzyme interactions [53]. However, other studies [65] suggest that Tyr355 may govern the stereospecificity of PGHSs toward NSAIDs and may play a role in a negative allosteric effect of AA in PGHS-1. The several other residues within the COX active site, besides Arg120, have been studied by mutagenesis in both isozymes [32,33,57,58]. Three regions of the active site have been studied extensively: Val349 near the apical portion of AA; Ser530, Tyr348, and Trp387 near the central portion of AA; Phe518, Leu531, and Gly533 near the ␻-end of the substrate. Mutations at three of these sites (Val349, Ser530, and Gly533; see Fig. 6) seem to reveal a great deal about the effects of substrate–enzyme interactions on catalysis. Val349 is situated between the carboxylate and middle half of AA, at the point where the substrate bends into a cul-de-sac off the main COX channel. This valine appears to be important in positioning AA for conversion to PGG2 [59]: the V349A mutant in ovine PGHS-1 and human PGHS-2 forms 11R-HPETE as the major product, not PGG2 [59]. The

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structural interpretation is that the AA conformer in these mutants has such an unfavorable stereochemistry in the region around carbons 9–11 that the 11R-hydroperoxy radical on AA can not attack carbon 9 to form the endoperoxide bridge. Hence, the reaction often aborts to form a predominance of 11R-HPETE by-product. The mutation of Val349 to other amino acids also has an impact on the formation of PGG2 and 15R/S-HPETE by-products [57,58]. In the penultimate step of PGG2 formation, a second molecule of oxygen inserts into the substrate at carbon 15 to create a 15S-hydroperoxy product. Moreover, wild-type PGHS-1 and -2 make predominantly the 15S-HPETE stereoisomer, as well as the 11R-HPETE by-product [57,58]. The replacement of Val349 with leucine in ovine PGHS-1 eliminates all 11-HPETE formation, but increases the formation of the 15R/S-HPETE by-products as an equimolar mixture [58]. When Schneider et al. [57] replaced valine with isoleucine at residue 349 in PGHS-1 or -2, they observed a marked shift in product stereochemistry at carbon 15 towards the R stereoisomer for both PGG2 and 15-HPETE. Thus, Val349 may play a subtle role in maintaining the proper stereochemistry of the substrate during O2 addition at carbon 15. Ser530, the site of acetylation by aspirin [42,66,67], has a close and intimate interaction with AA at carbon 13 [31,58]. While not essential for catalysis, Ser530 may help position AA with respect to the Tyr385 for hydrogen abstraction [31,60] at carbon 13, as well as for subsequent oxygen addition at carbon 11 (Fig. 6). When human PGHS-2 is acetylated by aspirin, substrate turnover still occurs, but 15R-HPETE is produced [67,68]. The additional atoms at position 530 clearly perturb the AA conformation so that oxygen insertion at carbon 11 is not possible after hydrogen abstraction. Moreover, molecular oxygen now adds to carbon 15, possibly from the side opposite to that seen in the native enzyme, to form eventually 15R-HETE. Acetylation of Ser530 in PGHS-1 completely inactivates the enzyme, suggesting that the addition of three nonhydrogen atoms completely blocks the binding of AA. As the COX active site in PGHS-2 is larger than in PGHS-1 [29,30], isoform 2 can accommodate the larger acetyl group without inactivating. When Ser530 in PGHS-1 is instead mutated to a threonine, the enzyme is active, but now behaves like aspirin-acetylated PGHS-2: 15R-HPETE is formed almost exclusively instead of PGG2 [58]. Xiao et al. [60] suggested that structural alterations at Ser530, arising from mutation or acetylation, flip the conformation of bound AA such that the 13proR hydrogen is abstracted instead of the 13proS. However, Schneider and Brash [69] clearly confirm that the 13proS hydrogen is still abstracted in aspirin-acetylated human PGHS-2. Thus, the perturbations in AA conformation that lead to 15R-HPETE formation are still unknown. Interestingly, Schneider et al. [57] found that replacing Ser530 in PGHS-2 with larger amino acids (e.g. threonine and methionine) resulted in an almost complete shift in product stereochemistry at carbon 15 towards the R stereoisomer in PGG2 . Hence, the size and stereochemistry of the amino acid side chain at position 530 plays a critical role in determining the stereochemistry of O2 addition at carbon 15. At the very end of the fatty acid binding pocket, Gly533 (Fig. 6) abuts against the very last two carbons of AA in PGHS-1 [31]. Intriguingly, the mutation of this residue in ovine PGHS-1 and human PGHS-2 markedly decreases substrate turnover [58,62,66]. The superimposed structures of the fatty acids DHLA, EPA, and LA [32,33] clearly show that the region of the COX active site between Ser530 and Gly533 tends to conform the ␻-end of the substrate into a particular stereochemistry (Figs. 6 and 7a). Mutations at or near Gly533

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must then perturb the conformation of the substrate away from the catalytically competent conformer [31,58,62]. Thus, it seems that the positioning of the ␻-end of a fatty acid substrate is a critical factor in its turnover [32,33]; if the tail of the substrate is not bound properly, then the appropriate hydrogen cannot be readily abstracted. On the other hand, the COX active site surrounding the carboxylate end of the fatty acids seems to accommodate mutations better without a major loss of COX activity [32,33]. This region of the active site may help compensate for any steric strain arising from the positioning of the substrate’s ␻-end for turnover.

8. Mechanistic implications for the general COX reaction The accumulating wealth of information derived from structural and mutagenic studies has highlighted some of the important steps in PGG2 formation. At least, four key features of the COX reaction scheme (Fig. 8) have been identified by biochemical evidence and seen in crystal structures. First, the arachidonyl carboxylate interacts with Arg120 [28–31,42,43]. Second, the 13proS hydrogen of AA is appropriately positioned for abstraction by a radical on Tyr385 [1,3,70]. The middle of the AA molecule is positioned in a space suitable for the formation of the endoperoxide bridge between carbons 11 and 9 [31,39]. Finally, the end of AA extends into in a side pocket of the channel around Gly533, which is very sensitive to mutation [62,66]. When AA is appropriately positioned such that the radical on Tyr385 can abstract the 13proS hydrogen from AA, there is the facile formation of the 11R-peroxyl radical achieved via O2 addition. In the next step, the 11R-peroxyl radical attacks carbon 9 to form the endoperoxide, resulting in the isomerization of the radical to carbon 8. However, the extended orientation exhibited by AA doesn’t allow for ring closure between carbon 8 and carbon 12 at this stage of the reaction because the distance between these carbons is ∼5 Å (Fig. 8). Therefore, a major reconfiguration of the substrate must occur concomitant with or immediately following formation of the endoperoxide bridge. This hypothetical conformational transition would involve a significant movement of the ␻-end towards the carboxyl half of the substrate. With the loss of the carbon 11/carbon 12 double bond as the 11R-hydroperoxyl moiety is formed, local conformational freedom increases for its attack on carbon 9. The 11R-hydroperoxyl radical would then swing “over” carbon 8 for an R-side attack on carbon 9 through the rotation about the carbon 10/carbon 11 bond, which brings carbon 12 closer to carbon 8 for the ring closure (Fig. 8). As carbons 13–20 are now repositioned, carbon 15 becomes optimally placed for addition of the second molecule of O2 . In the final step, the 15S-peroxyl radical is aligned below Tyr385 for donation of the radical to complete cyclooxygenase catalysis. Formation of PGG2 , 11R-HPETE, and 15S-HPETE by native PGHS-1 and -2 is always initiated by abstraction of the 13proS hydrogen. Because of the nonnative stereochemistry, the formation of 15R-HPETE was suggested to be initiated by removal of the 13proR hydrogen [60]. This hypothesis is weakened, however, by the recent work by Schneider and Brash [69] which shows that 15R-HPETE formation also arises from abstraction of the 13proS hydrogen. The work of Schneider et al. [57] also suggests that the formation of the R stereoisomer occurs due to altered substrate stereochemistry around carbon 15 at the time of O2 addition. As little conformational variation is seen in the PGHS-1 and -2 crystal

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structures, subtle, but distinct dynamic transitions in enzyme structure must be occurring to account for the different substrate conformers in the native and mutant forms of PGHS.

9. COX inhibition by NSAIDs As there are a number of recent and detailed reviews of this topic [52,71,72], only a summary of the major highlights NSAID inhibition in the PGHS isoforms is given. NSAID binding to the COX active sites of PGHS-1 and -2 have been studied extensively, and a number of crystal structures of NSAID/PGHS complexes are available [28–30,42,43]. NSAIDs can be subdivided into two classes of drug behavior: (a) classical, “isoform nonspecific” NSAIDs and (b) COX-2 inhibitors. All the classical NSAIDs inhibit both PGHS-1 and -2 but many tend to bind more tightly to PGHS-1 [71,72]. In contrast, COX-2 inhibitors have been designed to exhibit many-fold higher selectivity toward PGHS-2 [71,72]. Second, while all NSAIDs compete with arachidonate for the COX active site, each NSAID exhibits one of three general modes of inhibition [71,72]: (a) rapid, reversible binding (e.g. ibuprofen); (b) rapid, lower affinity reversible binding followed by time-dependent, higher affinity, slowly reversible binding (e.g. flurbiprofen); or (c) rapid, reversible binding followed by covalent modification (as in the acetylation of Ser530 by aspirin). The structural basis for time-dependent inhibition is not well-defined and may be different for different drugs (see below). The kinetic differences in NSAID inhibition have made simple comparisons of drug interactions with PGHS-1 versus PGHS-2 difficult, particularly in vitro.

10. The structural basis of NSAID binding and selectivity Fig 9a illustrates the basic features of classical NSAID binding to PGHS-1 and -2. The drugs tend to bind in the upper part of the COX channel between Arg120 and Tyr385. Structurally, the classical acidic NSAIDs (e.g. profens and fenamates) interact with Arg120 in both PGHS isozymes [52,72], where intimate hydrogen bonding or electrostatic interactions provide a major portion of the binding energy and selectivity. This is not always the case in PGHS-2 and is the basis for enhanced selectivity of the new “COX-2” selective drugs (see below). The remaining drug protein interactions tend to be hydrophobic [52,72] except for potential hydrogen bonding to Ser530. The physical basis for time-dependent inhibition in the PGHS isozymes is also not well understood. Indeed, the appearance of time-dependent inhibition seems to vary among inhibitor classes, and even between drugs within a given class. For example, flurbiprofen inhibits PGHS-1 by a time-dependent, pseudo-irreversible mechanism, but ibuprofen, a close chemical relative, does not [73]. Thus, some facet of the drug’s interaction with PGHS induces a conformational transition to a tight-binding EI∗ state [52,72]. For most acidic, time-dependent NSAIDs, interaction with Arg120 appears to be required [53,63]. Nonetheless, structural comparisons between the known PGHS-drug structures have yet to reveal the physical basis for time-dependent inhibition. The differences between classical NSAIDs and COX-2 inhibitors can be rationalized to some extent on slight differences between the COX active sites of PGHS-1 and -2.

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Fig. 9. A view of NSAIDs binding to the COX active site. Flurbiprofen (light blue) is bound in the COX active site channel in ovine PGHS-1 [28]. Residues Ile434 (yellow), His513 (green), Phe518 (purple), and Ile523 (yellow), are displayed as space filling (a). The COX-2 inhibitor SC-588 (light blue) is bound in the COX active site channel of mouse PGHS-2 [30]. Residues Val434 (yellow), Arg513 (green), Phe518 (purple), and Val523 (yellow) are displayed as space filling. The phenylsulfonamide group of SC-588 extends into the side pocket made accessible by Val523 and interacts with Arg513. Access to the side pocket is made easier by the I434V change in PGHS-2, which then allows Phe518 to move out of the way when COX-2 inhibitors bind in this pocket (b).

Substitution of Ile523 in PGHS-1 with Val523 in PGHS-2 results in a small side pocket becoming more accessible from the active site channel which, thus, appreciably increases the volume of the COX active site [29,30]. This change is compounded by the substitution of Ile434 in PGHS-1 with Val434 in PGHS-2, within the second shell of amino acids surrounding the COX active site. This substitution further increases the effective size of the active site channel by enhancing the local mobility of side chains within the side pocket. The combination of these two differences at positions 523 and 434 in PGHS-2 allows a movement of Phe518 that permits access to the polar side pocket (Fig. 9b). The larger main channel combined with this “side pocket” increases the volume of the PGHS-2 NSAID binding site by about 20% over that in PGHS-1 [29,30]. This extra volume is a structural feature exploited by most COX-2 inhibitors. When access to this side pocket is restricted by mutating Val523 to an isoleucine, PGHS-2 is no longer differentially sensitive to these inhibitors [74,75]. Conversely, an I523V mutation in PGHS-1 increases its affinity for COX-2 inhibitors [76]. The larger effective size of the central channel in the PGHS-2 may also preferentially reduce steric and ionic crowding by the charged Arg120 in PGHS-2 and thus, may enhance the binding of nonacidic NSAIDs to PGHS-2. The substitution of His513 in PGHS-1 with arginine in PGHS-2 also alters the chemical environment of the side pocket by placing a stable positive charge at its center [30]. This arginine seemingly interacts with polar moieties entering the pocket. For example, the interactions between Arg513 and the 4-methysulfonyl or 4-sulfonamoylphenyl substituents of diarylheterocyclic COX-2 inhibitors give rise to the time-dependent inhibition displayed

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by this class of inhibitors [76]. In combination with the I523V mutation, an H513R mutant of PGHS-1 becomes much more sensitive to COX-2 inhibitors [76]. The new COX-2 inhibitors exhibit time dependent, pseudo-irreversible inhibition towards human PGHS-2 and murine PGHS-2, while they inhibit PGHS-1 by a rapid, competitive and reversible mechanism (see reviews [52,72]). This is surprising as no effort was made to design in this behavior. As mentioned above, time-dependent inhibition of PGHS-2 by COX-2 inhibitors containing sulfonamide or methylsulfoxide moieties apparently arises from their interaction with Arg513 [30,76]. Curiously, inhibition by the methylsulfoxide inhibitor NS-398, an early lead compound, appears to depend on interaction with Arg120, but not with Arg513. NS-398 binds in the PGHS-2 active site similarly to acidic inhibitors and inhibits the R120E and R120Q PGHS-2 mutants only competitively [54,64]. Hence, “rational drug design” is still a trial and error procedure with a bit of serendipity added in.

11. Conformational transitions and membrane interactions Research over the last 20 years has made it clear that the PGHS is not a static globular protein, but a structurally dynamic molecule whose flexibility is integral to its activities. Not only does PGHS undergo conformational changes following binding of heme, fatty acid substrates, and NSAIDs, but also the conformational behavior is subtly, clearly different between PGHS-1 and -2. Some of the earliest evidence for global flexibility within the PGHS molecule comes from studies examining the protease sensitivity of PGHS-1. The apo-enzyme is selectively cleaved by trypsin at a single site, Arg277, to produce 33 and 38 kDa fragments [77,78]. In contrast, the addition of heme, NSAIDs, or arachidonate protects against proteolysis. The phenomenon of time-dependent inhibition also provides credible evidence for local conformation changes in PGHS [1,72] between a freely reversible enzyme–ligand complex EI and a tight-binding, slowly reversible EI∗ complex. The discovery of COX-2 selective inhibitors has revealed a new, and intriguing twist to this inhibitory phenomenon [72,74,75]: these new NSAIDs display time-dependent inhibition towards COX-2, but freely reversible inhibition of COX-1. Conformational models are now being invoked to explain this COX-2 selective time-dependent inhibition [65,79]. Intuitively, it seems likely that some conformational change in PGHS would be required to stabilize a tight-binding NSAID complex. More recently, the crystal structures of ovine PGHS-1 [28,31,43,80], murine PGHS-2 [20,34], and human PGHS-2 [29] highlight the implicit need for major conformational changes to explain ligand entry to (or exit from) the COX active site. All fatty acid substrates and NSAIDs must enter the COX active site through the MBD. The narrow aperture within the COX channel (Fig. 4) effectively buries all bound ligands within the catalytic domain [31,43]. This clearly infers that the MBD must undergo significant conformational changes during substrate entry and product exit. Recent studies [65,79] have suggested that the reorganization of hydrogen-bonding networks within the MBD [65] may play an active role in substrate binding, catalytic efficiency, and inhibition by NSAIDs. Despite the growing wealth of structural information on the PGHS isoforms, the crystals structures do not exhibit major ligand-induced conformational changes. This does not mean that all these structures are identical, but simply that the minor structural differences

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observed could be ascribed to local perturbations induced by the individual ligand used for co-crystallization. For example, RS57067 displaces Arg120 of PGHS-2 about 2.3 Å [29], but binding of two other time-dependent inhibitors, the chemically related RS104897 and flurbiprofen do not [29,30]. Minor structural variations are also observed for various NSAID and fatty acid substrate co-crystals, but no compelling evidence for a time-dependent inhibitor-specific conformation of either PGHS isozyme has been obtained from comparisons of the crystal structures. Why is only a single major conformation for the PGHS isoforms observed in crystals? The conformation of the COX active site seen in the various crystal structures must be quite stable. This raises the possibility that at least two conformations of the enzyme exist in equilibrium: a relatively unstable ligand-free conformation and a much more stable ligand-bound conformation. To this dilemma of conformation transitions in PGHS, one must also add the fact that PGHS is an integral membrane protein. The crystallographic analyses of PGHS, as well as many of the biochemical and pharmacological experiments, have required the isolation of the enzyme from the membrane and its solubilization by detergents. It is possible that the equilibrium between the different conformations in detergent-solubilized PGHS deviates from that found in the membrane-bound enzyme. Crystal structures of PGHS-1 and -2 have also been obtained without ligands [30] or with competitive inhibitors [80]. What is intriguing is that these structures are essentially indistinguishable from the structures of PGHS when complexed with time-dependent inhibitors, like flurbiprofen or COX-2 inhibitors. This may imply that the crystallization of detergent-solubilized PGHS promotes the structural transition to an EI∗ conformation. Two important questions then need to be resolved: (1) what are the structural relationships between the EI∗ conformation and the productive ES conformation and (2) does the conformational equilibrium markedly differ in the membrane-bound enzyme? Raising the issue of membrane interactions in PGHS, the domain organization of PGHS-1 and -2 suggests design implications for the COX enzymology and one source for the conformational transitions. As mentioned previously, the catalytic domain shares a great deal of structural homology with mammalian POXs [28,37], which are soluble proteins. From an evolutionary prospective, an ancestral POX underwent two distinct changes to create PGHS: (1) the formation of an interior channel for the COX reaction and (2) the acquisition of the MBD. The helices of the MBD form the opening to the first half of the hydrophobic COX channel leading into the interior of the catalytic domain [28] (Fig. 3). It is this interface between the MBD and the catalytic domain that creates the aperture in the COX active site. Thus, a ligand must pass through the MBD before entering the catalytic domain. This suggests that substrate binding to PGHS might occur in three distinct steps: initial ligand recognition at the channel mouth, entry into the COX channel, specific tight binding in the upper portion of the COX channel. Similar structural transitions have also been proposed to occur during NSAID binding [79]. Interestingly, the region of highest sequence divergence between mature PGHS-1 and -2 is the MBD [8,17]. How these conformational transitions are controlled and impact substrate and NSAID binding are unanswered. As yet, no hydrogen-bond network, within the MBD or across the MBD–catalytic domain interface, has been identified as the actual conformational switch for aperture opening and ligand entry. Identifying the EI∗ state might be easier. In the active PGHS, AA is rapidly converted to PGG2 and released. Oxygenation leads to a

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conformational rearrangement of AA chain within the active site as the cyclopentane ring and endoperoxide bridge form [31]. This structural transition may disrupt a hydrogenbonding network within the COX channel and facilitate release of the product PGG2 . Time-dependent NSAIDs easily bind within the COX channel, but may not be able to trigger the required conformational change needed for facile release. Thus, time-dependent inhibition may be a serendipitous outcome of a catalytic mechanism evolved in PGHS to ensure fidelity of the COX reaction. Nonetheless, identification and characterization of the important conformational transitions in PGHS remain elusive, but these structural events will have a major impact on our understanding of how NSAIDs interact with PGHS.

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