Structure and Dynamics of Cyclooxygenase Catalysis and Inhibition

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 DESCRIPTION (provided by applicant): Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid to generate prostaglandins and are inhibited by aspirin, nonselective nonsteroidal anti-inflammatory drugs (NSAIDs), and COX-2 selective diarylheterocyclic based drugs (coxibs). COX-1 and COX-2 are membrane-associated homodimers that bind to one leaflet of the lipid bilayer via a membrane-binding domain comprised of amphipathic helices. As a consequence, the enzymes have traditionally required the presence of detergents to maintain the protein in a stable and active form during functional and structural characterization. A new paradigm has emerged with respect to COX catalysis and regulation. In this model, COX functions as a conformational heterodimer with one monomer active at a given time. Common dietary FAs and nonselective NSAIDs bind to one monomer of the homodimer to modulate the oxygenation of substrates in the other monomer through an allosteric/catalytic couple. The molecular mechanism governing crosstalk between monomers is not known and static pictures derived from crystal structures of COX do not provide any insight into the conformational motions responsible for this dynamical interplay. We surmise that detergent binding and the restricted confines of the crystal lattice have masked these conformational motions. Hence, a new approach to study COX catalysis in solution and in the absence of detergent is needed. We propose to couple the use of nanodisc-reconstituted COX-2 with site- directed spin-labeling ESR spectroscopic technologies to characterize the protein conformational dynamics associated with COX catalysis and inhibition. The objectives are to identify the conformational motions responsible for: a) ligand access to the cyclooxygenase channel and b) communication between monomers. Understanding the molecular basis of how the binding of different ligands induces conformational motions responsible for crosstalk between monomers bridges the gap between the static information derived from COX crystal structure analysis and the dynamical character of COX as it relates to the modulation of COX function.