The membrane binding domains of prostaglandin endoperoxide H synthases 1 and 2. Peptide mapping and mutational analysis.
Prostaglandin endoperoxide H synthases 1 and 2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs. Both isozymes are integral membrane proteins but lack transmembrane domains. X-ray crystallographic studies have led to the hypothesis that PGHS-1 and -2 associate with only one face of the membrane bilayer through a novel, monotopic membrane binding domain (MBD) that is comprised of four short, consecutive, amphipathic alpha-helices (helices A-D) that include residues 74-122 in ovine PGHS-1 (oPGHS-1) and residues 59-108 in human PGHS-2 (hPGHS-2). Previous biochemical studies from our laboratory showed that the MBD of oPGHS-1 lies somewhere between amino acids 25 and 166. In studies reported here, membrane-associated forms of oPGHS-1 and hPGHS-2 were labeled using the hydrophobic, photoactivable reagent 3-trifluoro-3-(m-[(125)I]iodophenyl)diazirine, isolated, and cleaved with AspN and/or GluC, and the photolabeled peptides were sequenced. The results establish that the MBDs of oPGHS-1 and hPGHS-2 reside within residues 74-140 and 59-111, respectively, and thus provide direct provide biochemical support for the hypothesis that PGHS-1 and -2 do associate with membranes through a monotopic MBD. We also prepared HelA, HelB, and HelC mutants of oPGHS-1, in which, for each helix, three or four hydrophobic residues expected to protrude into the membrane were replaced with small, neutral residues. When expressed in COS-1 cells, HelA and HelC mutants exhibited little or no catalytic activity and were present, at least in part, as misfolded aggregates. The HelB mutant retained about 20% of the cyclooxygenase activity of native oPGHS-1 and partitioned in subcellular fractions like native oPGHS-1; however, the HelB mutant exhibited an extra site of N-glycosylation at Asn(104). When this glycosylation site was eliminated (HelB/N104Q mutation), the mutant lacked cyclooxygenase activity. Thus, our mutational analyses indicate that the amphipathic character of each helix is important for the assembly and folding of oPGHS-1 to a cyclooxygenase active form.
Spencer
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Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Thuresson
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E
Otto
J C
JC
Song
I
I
Smith
T
T
DeWitt
D L
DL
Garavito
R M
RM
Smith
W L
WL
D000595:Amino Acid Sequence; D000818:Animals; D001389:Azirines; D001665:Binding Sites; D019556:COS Cells; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism; D019920:Photoaffinity Labels; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012756:Sheep; D012995:Solubility; D014162:Transfection
D001389:Azirines; D007527:Isoenzymes; D008565:Membrane Proteins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D010544:Peroxidases; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases