A computational protocol for the integration of the monotopic protein prostaglandin H2 synthase into a phospholipid bilayer

Abstract

Prostaglandin H2 synthase (PGHS) synthesizes PGH2, a prostaglandin precursor, from arachidonic acid and was the first monotopic enzyme to have its structure experimentally determined. Both isozymes of PGHS are inhibited by nonsteroidal antiinflammatory drugs, an important class of drugs that are the primary means of relieving pain and inflammation. Selectively inhibiting the second isozyme, PGHS-2, minimizes the gastrointestinal side-effects. This had been achieved by the new PGHS-2 selective NSAIDs (i.e., COX-2 inhibitors) but it has been recently suggested that they suffer from additional side-effects. The design of these drugs only made use of static structures from x-ray crystallographic experiments. Investigating the dynamics of both PGHS-1 and PGHS-2 using classical molecular dynamics is expected to generate new insight into the differences in behavior between the isozymes, and therefore may allow improved PGHS-2 selective inhibitors to be designed. We describe a molecular dynamics protocol that integrates PGHS monomers into phospholipid bilayers, thereby producing in silico atomistic models of the PGHS system. Our protocol exploits the vacuum created beneath the protein when several lipids are removed from the top leaflet of the bilayer. The protein integrates into the bilayer during the first 5 ns in a repeatable process. The integrated PGHS monomer is stable and forms multiple hydrogen bonds between the phosphate groups of the lipids and conserved basic residues (Arg, Lys) on the protein. These interactions stabilize the system and are similar to interactions observed for transmembrane proteins.

FIGURE 1

The secondary and tertiary structure of ovine sp. prostaglandin H2 synthase-1 dimer. A side view of PGHS is drawn in panel A and the different domains are labeled. The EGF-like domains are colored red, the membrane binding domain yellow, and the catalytic domains, blue and gray. Flurbiprofen and the heme cofactor are colored green and pink, respectively, in the left-hand (gray) monomer. All views are with respect to the postulated plane of the membrane, which is shown as a dark line. The view from underneath PGHS is drawn in panel B and the four different helices (A–D) of the MBD are labeled.

FIGURE 2

Snapshots of the monomeric PGHS-1 system at different times. The nitrogen atoms of the choline groups of the POPC lipids are drawn as blue spheres to allow easy identification of the lipid-water interface.

FIGURE 3

The atomic densities, ρ(z), as a function of z for the membrane binding domain (solid line), the whole enzyme (dotted line), and the phospholipid bilayer (dashed line) for both PGHS-1 and PGHS-2 show the integration of the protein into the bilayer. Panels A–C and D and E show the progressive integration of PGHS-1 and PGHS-2, respectively, at t = 0, t = 5 ns, and t = 14.75 ns. The value ρ(z) is averaged over a 250 ps window and is measured in kg m⁻³.

FIGURE 4

The number of close contacts as a function of time, c(t), for each of the four systems studied (see Table 1), reaches a plateau after ∼5 ns.

FIGURE 5

Atomic densities, ρ(z), as a function of z for the MBD (solid line), the whole enzyme (dotted line), and the phospholipid bilayer (dashed line) for the restrained integration of aPGHS-1. Densities are plotted at different times: t = 0 (panel A), t = 5 ns (panel B), and t = 14.75 ns (panel C). The value ρ(z) is averaged over a 250 ps window and is measured in kg m⁻³.

FIGURE 6

The number of close contacts as a function of time, c(t), for the restrained integration of apo PGHS-1. The restrained and unrestrained simulation are drawn with a solid line and a broken line, respectively.

FIGURE 7

The root mean-square deviations as a function of time for each of the four systems studied. The differences between the RMSDs for the different systems shown in panel A are reduced by fitting on each domain separately (B), thereby removing any interdomain motion. Either RMSD measure for all four systems is <3 Å throughout and therefore their structures remain similar to their x-ray crystallographic structures.

FIGURE 8

The force applied to PGHS for the bound (dashed lines) systems and the unbound (solid lines) systems, showing how a significantly larger force is required to move the bound system the same distance as the unbound system. Due to the fluctuations in the force, each bound and unbound case was repeated twice.

FIGURE 9

Snapshots from a steered molecular dynamics simulation as PGHS is pulled free of the phospholipid bilayer at three different values of the displacement, d. Note the deformation of the lipid bilayer.

FIGURE 10

Multiple-alignment of PGHS-1 and PGHS-2 primary sequences (MBD region only). Note that the first two sequences are those with x-ray crystallographic structures and are those studied in this article. Basic residues (W,Y) and aromatic amphipathic residues (R,K) are colored purple and yellow, respectively.

FIGURE 11

The distribution of basic and aromatic amphipathic residues on the membrane binding domain (MBD). oPGHS-1 is on the left, mPGHS-2 is on the right. Basic residues (Arg, Lys) are shown in blue and aromatic amphipathic residues (Trp, Tyr) are shown in yellow. Aromatic and amphipathic residues have been shown to interact with individual phospholipids and stabilize transmembrane proteins in membranes.

FIGURE 12

An example of a typical interaction between an arginine of cPGHS-1 and several phospholipids. Panel A shows Arg-114 of cPGHS-1 forming four hydrogen bonds, labeled i–iv, with two separate POPC lipids. The hydrogen-bonding distances for these bonds are plotted in panels B and C. The bonds persist for up to 10 ns and the guanidinium group bonds to different oxygens on the phospholipids. Hydrogen bonds are indicated by a dashed line and each is labeled. The lipids are drawn without hydrogens for clarity and only the MBD is drawn and the different helices are labeled.

FIGURE 13

An example of a typical interaction between a lysine of cPGHS-2 and a phospholipid. Panel A shows Lys-114 of cPGHS-1 forming a single hydrogen bond, labeled (v), with the phosphate group of a POPC lipid. This hydrogen bond is intermittent and persists for no longer than 2 ns (panel B). The lipids are drawn without hydrogens for clarity and only the MBD is drawn and the different helices are labeled.

FIGURE 14

The total number, h(t), of hydrogen bonds between the MBD and the lipid bilayer (solid line) for the four PGHS systems studied (see Table 1). The cumulative number of hydrogen bonds involving arginine (dashed line) and lysine (dotted line) are also plotted. A hydrogen bond is assumed to have formed if the distance between donor and acceptor is <3.5 Å and the deviation is <30°. The total number of hydrogen bonds for apo-PGHS-1 and -2 is plotted in panels A and B, and for h(t) for flurbiprofen-bound PGHS-1 and -2 is plotted in panels C and D.