title: Ca2+-signaling cycle of a membrane-docking C2 domain.
abstract: The C2 domain is a Ca2+-dependent, membrane-targeting motif originally discovered in protein kinase C and recently identified in numerous eukaryotic signal-transducing proteins, including cytosolic phospholipase A2 (cPLA2) of the vertebrate inflammation pathway. Intracellular Ca2+ signals recruit the C2 domain of cPLA2 to cellular membranes where the enzymatic domain hydrolyzes specific lipids to release arachidonic acid, thereby initiating the inflammatory response. Equilibrium binding and stopped-flow kinetic experiments reveal that the C2 domain of human cPLA2 binds two Ca2+ ions with positive cooperativity, yielding a conformational change and membrane docking. When Ca2+ is removed, the two Ca2+ ions dissociate rapidly and virtually simultaneously from the isolated domain in solution. In contrast, the Ca2+-binding sites become occluded in the membrane-bound complex such that Ca2+ binding and dissociation are slowed. Dissociation of the two Ca2+ ions from the membrane-bound domain is an ordered sequential process, and release of the domain from the membrane is simultaneous with dissociation of the second ion. Thus, the Ca2+-signaling cycle of the C2 domain passes through an active, membrane-bound state possessing two occluded Ca2+ ions, one of which is essential for maintenance of the protein-membrane complex.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado at Boulder 80309-0215, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Nalefski
authors.forename: M M
authors.initials: MM
authors.lastname: Slazas
authors.forename: J J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D054467:Phospholipases A2; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
chemical_list: D008081:Liposomes; D010713:Phosphatidylcholines; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium