Arachidonic acid and nonsteroidal anti-inflammatory drugs induce conformational changes in the human prostaglandin endoperoxide H-2 synthase-2 (cyclooxygenase-2)

By using the technique of site-directed spin labeling combined with EPR spe ctroscopy, we have observed that binding of arachidonic acid and nonsteroid al antiinflammatory drugs induces conformational changes in the human prost aglandin endoperoxide H-2 synthase enzyme (PGHS-2), Line shape broadening r esulting from spin-spin coupling of nitroxide pairs introduced into the mem brane-binding helices of PGHS-2 was used to calculate the inter-helical dis tances and changes in these distances that occur in response to binding var ious ligands, The inter-residue distances determined for the PGHS-2 holoenz yme using EPR were 1-7.9 Angstrom shorter than those of the crystal structu re of the PGHS-8 holoenzyme, However, inter-helical distances calculated an d determined by EPR for PGHS-S complexed with arachidonic acid, flurbiprofe n, and CS-58125 were in close agreement with those obtained from the cognat e crystal structures. These results indicate that the structure of the solu bilized PGHS-S holoenzyme measured in solution differs from the crystal str ucture of PGHS-2 holoenzyme obtained by x-ray analysis. Furthermore, bindin g of ligands induces a conformational change in the holo-PGHS-2, converting it to a structure similar to those obtained by x-ray analysis. Proteolysis protection assays had previously provided circumstantial evidence that bin ding of heme and non-steroidal anti-inflammatory drugs alters the conformat ion of PGHS, but the present experiments are the first to directly measure such changes. The finding that arachidonate can also induce a conformationa l change in PGHS-2 was unexpected, and the magnitude of changes suggests th is structural flexibility may be integral to the cyclooxygenase catalytic m echanism.