A Computational Protocol for the Integration of the Monotopic Protein Prostaglandin H2 Synthase into a Phospholipid Bilayer

INTRODUCTION

Monotopic proteins are integral membrane proteins but, unlike transmembrane proteins, their polypeptide chains do not cross the phospholipid bilayer (1). To date only four monotopic proteins have had their structures experimentally determined: prostaglandin H2 synthase (PGHS) (2), squalcne-hopene cyclase (3). monoamine oxidase (4). and fatty acid amide hydrolase (5). There is tremendous academic and industrial interest in monotopic proteins; for example, all four enzymes for which structures exist are important pharmaceutical drug targets.

PGHS (EC No. 1.14.99.1) catalyses the conversion of arachidonic acid, a 20-carbon fatty acid, to prostaglandin H^sub 2^ (PGH^sub 2^). the precursor of the prostaglandin class of local hormones. The reaction proceeds within PGHS in two steps at spatially distinct active sites: arachidonatc and two molecules of oxygen are reacted together at the cyclooxygenase (COX) site to form prostaglandin G^sub 2^ (PGG^sub 2^). which is then reduced to PGH^sub 2^ at the peroxidase site (6). PGHS is often called COX due to its cyclooxygenase function. We will not discuss the structure, biochemistry, and inhibition of PGHS in detail as there are many excellent reviews (7-11 ).

PGHS is interesting because not only is it a monotopic protein but also two genes encode similar PGHS enzymes (11). Additional splice variants of the PGHS-1 isozyme have been proposed, potentially increasing this number (12). PGHS-1 is constitutively expressed and synthesizes prostaglandins involved in homeostasis, for example maintaining the mucosal lining of the stomach (13). PGHS-2 is induced and rat models have demonstrated that this enzyme is involved in local pain and inflammation responses (14.15). The PGHS enzymes have also been implicated in other human pathologies, for example in various cancers (10).

Nonsteroidal antiinflammatory drugs (NSAIDs), such as aspirin, ihuprofen, and flurhiprofen. hind within the cyclooxygenase active site, limiting the production of PGH^sub 2^ and therefore inhibiting the action of the enzyme. The ulcerogenic and renal side-effects of NSAIDs arc caused by inhibition of PGHS-1 and the majority of classical NSAIDs (e.g., aspirin and ibuprofen) are either not selective or inhibit PGHS-1. Consequently there has been a tremendous effort in the last decade to design an NSAID that is specific to PGHS-2 and therefore has reduced side-effects, while maintaining the desired analgesic and antiinfiammatory actions. This culminated in IW) in the introduction of several so-called COX-2 inhibitors, most notably celecoxib (Celebrex. Pfizer) and rofecoxib (Vioxx. Merck). Interest in these important clinical inhibitors has increased recently due to the withdrawal of rofecoxib in 2004 by its manufacturer because of a higher incidence of myocardial infarction during an extended clinical trial. The mechanism of this side-effect is not known. A recent population study by Hippisley-Cox and Coupland (16) suggested that other NSAIDs. including ibuprofen. may also suffer from the same side-effect, albeit to differing degrees.

Designing an isozyme-specific inhibitor is an extremely difficult task because the isozymes are structurally very similar. For example, the sequence similarity between PGHS-1 and PGHS-2 is 60-65% within the same species (10) and the root mean-square deviation ( RMSD) over the C^sub a^ atoms is 0.9-1.0 [Angstrom] when comparing the different sheep PGHS-1 and mouse PGHS-2 x-ray crystal structures. The PGHS active site, as defined by those residues in close contact with the bound substrate, is highly conserved with only a single amino acid difference (1523V) between isozymes. We will follow the convention of using the amino acid numbering of sheep PGHS-1. The less bulky side chain of valine compared to isolcucine permits ligands to access an additional side-pocket seen in the x-ray crystallographic structures of PGHS-2 (17). The COX-2 inhibitors exploit this pocket and mutagenesis experiments have demonstrated that this mutation is important in producing the selectivity of these new inhibitors (18-20).

The x-ray crystallographic structure of a monotopic protein is a static snapshot of the protein at cryogenic temperatures and contains little dynamical information. Kinetic studies have revealed that the dynamics of PGHS is important for its inhibition by NSAIDs (21.22). Studying the dynamics of PGHS-1 and PGHS-2 is likely to lead to additional insight beyond that gained when comparing static x-ray crystallography structures and therefore could inform future drug design. This study is the first step toward analyzing such dynamical differences between PGHS-1 and -2.

We use large-scale classical molecular dynamics (MD) to study the dynamics of this system. MD is constrained by the short timescales, typically tens of nanoseconds, that it can access relative to those of more general biological interest, but it allows insight to be gained into the dynamics (and therefore behavior) of these proteins that is not possible by experiment. Hypotheses may then be generated which can he tested experimentally in an iterative process.

It is not known what aspects of the system will be important in determining the dynamical differences (if any) between the PGHS isozymes and it is therefore prudent to include a phospholipid bilayer, since this forms many interactions with PGHS. Unfortunately, it is difficult to build atomistic models of monotopic proteins anchored to a membrane as, unlike transmembrane proteins, there are no clear transmembrane units (e.g., a-helices or a ß-barrel) with which to position the protein relative to the membrane. The purpose of this article is to outline a protocol for integrating a PGHS monomer into a phospholipid hilayer. We expect a PGHS dimer to integrate in the same way as the monomer. We shall present evidence to demonstrate that the protein is correctly inserted before making some concluding remarks.

METHOD

In this section we describe the structure of PGHS, define our protocol for integrating a PGHS monomer with a phospholipid bilayer, and provide details about the MD algorithm used.

Structure of prostaglandin H2 synthase

PGHS is situated on both the luminal side of the endoplasmic reticulum and on the nuclear membrane (23,24). X-ray crystallography indicates that the protein is a dimer, is mainly a-helical, and comprises 550-553 residues per monomer. Each monomer has three domains (see Fig. 1): an epidermal-growth factor (EOF)-like domain (residues 33-72), the catalytic domain (residues 117-586), and the membrane-binding domain (MBD, residues 73-116). A PGHS monomer is not biologically active, and therefore, if we drew any conclusions from the simulations of the monomer about the dynamical differences between the two isozymes. we would first have to first establish that the structure and dynamics of an integrated PGHS monomer are similar to that of an integrated PGHS dimer.

The MBD is composed of four a-helices, A, B, C, and D, the first three roughly forming three sides of a square with D connecting this motif to the catalytic domain. Picot et al. (2) hypothesized that these three short helices lie in the plane of the membrane near one interface, form numerous interactions with the top leaflet of the bilayer, and thereby bind the protein to the membrane. This was supported by surface plots of the hydrophohicity of the protein and comparison with other globular heme-containing peroxidases lacking the EGF and MBD domains, notably mammalian myeloperoxidase (10). The hypothesis was subsequently confirmed by a variety of photolabeling, mutagenesis (23,25), and fluorescent protein fusion experiments (24).

The hydrophohic substrate, arachidonic acid, is partitioned into the membrane and, it is assumed, enters the enzyme directly via the MBD. After cyclooxygenation, the product, PGG^sub 2^, is expelled from the COX site of PGHS and then binds to a peroxidase-active site where the final conversion to PGH^sub 2^ lakes place. Differences between PGHS-1 and PGHS-2 that may be exploited in the design of inhibitors could potentially be in any one of these steps. We assume here that it is most likely that differences will occur in the interactions between the drug and the enzyme: hence, we study flurhiprofen bound to the COX active site of PGHS-1 and -2. However, it is possible that other important differences may exist-for example, in the mechanism of entry of the substrate into each isozyme. This has been studied by Molnar et al. (26) using steered molecular dynamics.

Integration protocol

We will now describe the protocol used to integrate a monomer PGHS with a phospholipid bilayer. It was decided to integrate monomers rather than dimers to limit the size the system: however, we expect this approach to apply to PGHS dimers also. The protocol exploits the force exerted by the vacuum created when a number of lipids are removed from the bilayer directly beneath PGHS to integrate the enzyme rapidly into the membrane. This approach was first used by Nina et al. (27) to integrate the MBD into a small patch of lipids and we extend it here to an entire PGHS monomer. This has the advantage that we do not need any restraints on the protein and, by virtue of increased computer processor speeds and improved algorithms, we were able to evolve our models for 15 ns compared to the 1ns performed by Nina et al. (27).

The membrane plug-in to VMD1.8.3 (28) was used to generate a patch of bilayer comprising 214-234 1-palmitovl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipids, POPC being the most common constituent of the endoplasmic reticulum (29). The protein was oriented such that helices A, B. and C of the MBD were in the plane of the phospholipid hilayer. Defining the z axis as perpendicular to the phospholipid hilayer with its origin at the center of the bilayer (see Fig. 1). The number of lipids within 2 [Angstrom] of the protein was calculated as a function of z. The center of mass of the protein was then moved to a z coordinate (48-50 [Angstrom]) such that it was in close contact with 17 lipids.

Deciding how many lipids to remove is a packing problem: in principle, one should remove the number of lipids (11) equivalent to the cross-sectional area of the MBD to ensure the membrane is minimally perturbed. This, however, does not allow the enzyme to he positioned very close to the bilayer as the MBD does not fit well into the cavity produced due to discrete size effects. An additional six lipids were removed to allow the MBD to better lit the cavity and we expect the perturbation introduced by the integration of PGHS into the membrane to dominate any potential perturbation in the curvature of the membrane due to the removal of these additional lipids. Any effect is further reduced by using a large patch of POPC lipids.

Having removed these 17 POPC lipids to create the cavity beneath the protein, the protein was solvaled and then neutralized by adding counlerions; care w as taken to ensure no water molecules entered this cavity. The potential energy of the system was then minimized and the water and side chains relaxed before the system was thermalized up Io physiological temperature (310 K) over 0.5 ns. During the wanning, harmonic restraints in the z direction were applied to selected heavy alums within the headgroups of the phospholipids The magnitude of the restraints was decreased and removed before a Berendsen harostat was applied to maintain the system at 1 atm pressure. A Langevin thermostat was used to maintain the temperature at 310 K and production runs were 15 ns long. This duration is typical for current transmembrane protein simulations (30).

Systems studied

Table 1 lists the lour PGHS monomers studied and their respective Protein Data Bank (PDB) axles (31). The home group was included but the proteins were not glycosylated, as it has been shown that this is not necessary for their function (10).