Examination of the membrane binding domain of human cyclooxygenase-2 enzyme using site-directed spin labeling and electron paramagnetic resonance spectroscopy.

摘要

The widespread use of aspirin, ibuprofen, Celebrex®, Vioxx® and other non-steroidal anti-inflammatory drugs (NSAIDs) has called for a deeper understanding of the physiological systems in which they work. NSAIDs exerts their anti-inflammatory and analgesic effects through the inhibition of cyclooxygenase −1 and −2 (COX-1 and -2). The biological necessity for two COX isozymes is not fully understood as these enzymes have nearly identical catalytic properties. Several evidences support that the COX isozymes have different biological functions. The basis for the differential biological activities is not known, but may relate to differences in their membrane binding domains, which are poorly conserved.; Site-directed spin labeling (SDSL) is a powerful tool for determination of the membrane protein structure and the topology of protein binding to the lipid bilayer. 25 mutant enzymes that contained single reactive cysteines substituted for amino acids within the human COX-2 membrane-binding domain were constructed and spin labeled. The accessibility of each protein-bound spin label with freely diffusing oxygen as a non-polar paramagnetic reagent, and NiEDDA as a polar paramagnetic reagent, was measured using power saturation EPR spectroscopy. Our results show the accessibility parameter (П) for both oxygen and NiEDDA, has a periodicity of 3.6 consistent with an α-helical configuration. Furthermore, П_oxygen and П_NiEDDA both have the same period and phase. Our results indicate that both polar and non-polar paramagnetic relaxers do not access the amino acids located in the membrane-binding domain due to the very low values of П_oxygen and П_NiEDDA. This indicates that the membrane binding domain of hCOX-2 is located in the interfacial region of the membrane and is sandwiched between the main body of the 144 KDa COX dimer and the lipid bilayer. We have identified three amino acids that participate in anchoring the protein to the lipid bilayer. Human COX-2 is the first peripheral membrane protein mapped using site-directed spin labeling.; Another aspect of this dissertation was to study the conformational changes in the mouth of the cyclooxygenase channel. The purpose of this research was to determine whether the COX substrate and inhibitor binding sites are flexible, e.g., can be opened and closed. Ten cysteine double mutants located in the helices that form the opening of the cyclooxygenase active site and membrane binding of human COX-2 were constructed. The dipolar broadening in the presence and absence of NSAIDs, arachidonic acid and heme were compared in detergent. The same experiments were also performed in liposomes. The interspin distance measurements were done using Fourier convolution/deconvolution technique.; Our results indicated that binding of heme, arachidonic acid, and NSAIDs had no significant effect on the conformation of hCOX-2. However, a slight conformational change was observed in the structure of the enzyme in the presence and absence of the lipid bilayer.