Abstract
Prostaglandin endoperoxide H synthase-2 (PGHS-2), also called cyclooxygenase-2 (COX-2), converts arachidonic acid to PGH2. PGHS-2 is a conformational heterodimer composed of allosteric (Eallo) and catalytic (Ecat) subunits. Fatty acids (FAs) bind to Arg-120 of Eallo increasing to different degrees, depending on the FA, the Vmax of its Ecat partner. We report here that movement of helical residues 120–122 and loop residues 123–129 of Eallo underlies the allosteric effects of FAs and allosteric COX-2 inhibitors, including naproxen and flurbiprofen. An S121P substitution in both PGHS-2 monomers yields a variant (S121P/S121P PGHS-2) that has 1.7–1.8 times the Vmax of native PGHS-2 and is relatively insensitive to activation by FAs or inhibition by allosteric inhibitors. The S121P substitution in Eallo is primarily responsible for these effects. In X-ray crystal structures, the Cα atoms of helical residues 119–122 of S121P/S121P PGHS-2 are displaced from their normal positions. Additionally, the S121P/S121P PGHS-2 variants in which Pro-127 and Ser-541 are replaced by cysteines spontaneously forms Cys-127 to Cys-541 cross-links between monomers. This is unlike the corresponding native PGHS-2 variant and suggests that S121P substitutions also unhinge the loop involving residues 123–129. We conclude the following: (a) the region involving residues 120–129 of unoccupied Eallo tonically inhibits Ecat; (b) binding of an activating FA (e.g. arachidonic, palmitic, or oleic acid) to Eallo or an S121P substitution in Eallo repositions this region to increase Ecat activity; and (c) allosteric COX inhibitors act by preventing FA binding to Eallo and additionally by relocating Eallo residues to inhibit Ecat.
Highlights
Prostaglandin endoperoxide H synthase-2 (PGHS-2), called cyclooxygenase-2 (COX-2), converts arachidonic acid to prostaglandin H2 (PGH2)
Effects of Substitutions of Residues 120 –127 and Neighboring Residues of huPGHS-2—In a first set of experiments, we examined the effects of mutations in the huPGHS-2 dimer (a) near the C terminus of helix D, (b) in the loop downstream of helix D, (c) of Arg-44 which forms a hydrogen bond with Asp-125 of the loop, and (d) of Ser-541 and Ala-543 that neighbor this allosteric loop (Fig. 1 and Table 1)
As might be expected were the S121P substitution partially replacing the allosteric stimulation of huPGHS-2 by Fatty acids (FAs), Y385F S121P/Native huPGHS-2 was less sensitive to allosteric stimulation by palmitic acid (PA) than was the Y385F/S121P mutant (Table 5)
Summary
Effects of Substitutions of Residues 120 –127 and Neighboring Residues of huPGHS-2—In a first set of experiments, we examined the effects of mutations in the huPGHS-2 dimer (a) near the C terminus of helix D, (b) in the loop downstream of helix D, (c) of Arg-44 which forms a hydrogen bond with Asp-125 of the loop, and (d) of Ser-541 and Ala-543 that neighbor this allosteric loop (Fig. 1 and Table 1). Substitution of Arg-120 with alanine abrogates the allosteric effects of PA, oleic acid, and 20:19 that are not COX substrates (i.e. non-substrate fatty acids (nsFAs)) (Table 1) [9] This is because FAs do not efficiently bind to Eallo of R120A/R120A huPGHS-2. Mutant enzyme had somewhat reduced activity toward a variety of other substrates tested, including docosahexaenoic acid, linoleic acid, and 11,14-eicosadienoic acid; in the case of 11,14eicosadienoic acid, there is a significant increase in the Km value None of these latter three substrates efficiently bind to Eallo of native huPGHS-2 [11]. Given the large observed displacement of Arg-120 in the S121P/apo-crystal structure, we hypothesized that NSAIDs requiring an interaction with Arg-120 would not bind to S121P/ S121P muPGHS-2 To test this hypothesis, we employed a thermal shift assay to monitor the binding of FBP, IBP, naproxen, and celecoxib to native, R120A/R120A, and S121P/S121P muPGHS-2 variants (Fig. 5). FBP was relatively ineffective in preventing oxidant-induced cross-linking
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