Arachidonic acid and nonsteroidal anti-inflammatory drugs induce conformational changes in the human prostaglandin endoperoxide H2 synthase-2 (cyclooxygenase-2).
By using the technique of site-directed spin labeling combined with EPR spectroscopy, we have observed that binding of arachidonic acid and nonsteroidal anti-inflammatory drugs induces conformational changes in the human prostaglandin endoperoxide H(2) synthase enzyme (PGHS-2). Line shape broadening resulting from spin-spin coupling of nitroxide pairs introduced into the membrane-binding helices of PGHS-2 was used to calculate the inter-helical distances and changes in these distances that occur in response to binding various ligands. The inter-residue distances determined for the PGHS-2 holoenzyme using EPR were 1-7.9 A shorter than those of the crystal structure of the PGHS-2 holoenzyme. However, inter-helical distances calculated and determined by EPR for PGHS-2 complexed with arachidonic acid, flurbiprofen, and SC-58125 were in close agreement with those obtained from the cognate crystal structures. These results indicate that the structure of the solubilized PGHS-2 holoenzyme measured in solution differs from the crystal structure of PGHS-2 holoenzyme obtained by x-ray analysis. Furthermore, binding 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 binding of heme and non-steroidal anti-inflammatory drugs alters the conformation of PGHS, but the present experiments are the first to directly measure such changes. The finding that arachidonate can also induce a conformational change in PGHS-2 was unexpected, and the magnitude of changes suggests this structural flexibility may be integral to the cyclooxygenase catalytic mechanism.
Smith
T
T
Department of Biochemistry and the Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
McCracken
J
J
Shin
Y K
YK
DeWitt
D
D
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D016718:Arachidonic Acid / Q000494:pharmacology*; D001665:Binding Sites; D002460:Cell Line; D018360:Crystallography, X-Ray; D051546:Cyclooxygenase 2; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D008024:Ligands; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D011487:Protein Conformation; D018411:Spodoptera
D000894:Anti-Inflammatory Agents, Non-Steroidal; D007527:Isoenzymes; D008024:Ligands; D008565:Membrane Proteins; D016718:Arachidonic Acid; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases