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.

Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity

“…Finally, immediately prior to Ca 2+ affinity measurements, the protein was loaded onto a Chelex-100 resin column equilibrated with standard assay buffer containing 100 mM KCl, 20 mM N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 with KOH. Passage over this column removed all bound and free Ca 2+ from the protein sample, and replaced the purification buffer with standard assay buffer (18). The residual contaminating Ca 2+ concentration is submicromolar (18).…”

“…Ca 2+ binding to these sites is known to trigger a local structural change that increases the intrinsic emission of Trp 71 by 15%. The resulting fluorescence increase has been used previously to monitor the titration of sites I and II with two Ca 2+ ions, yielding a [Ca 2+ ] 1/2 of 14 ± 2 μM and a Hill coefficient of 1.7 ± 0.2 (6,18). Since the Hill coefficient significantly exceeds 1.0, there is strong positive cooperativity between the two adjacent sites (18).…”

“…The resulting fluorescence increase has been used previously to monitor the titration of sites I and II with two Ca 2+ ions, yielding a [Ca 2+ ] 1/2 of 14 ± 2 μM and a Hill coefficient of 1.7 ± 0.2 (6,18). Since the Hill coefficient significantly exceeds 1.0, there is strong positive cooperativity between the two adjacent sites (18). The [Ca 2+ ] 1/2 and Hill coefficient measured using the fluorescence assay agree closely with the values determined by direct Ca 2+ titration, validating the fluorescence method and confirming that the two binding sites contribute approximately equally to the fluorescence change (6,18).…”

“…Together they provide Ca 2+ coordination for up to four potential Ca 2+ -binding sites labeled sites I-IV; however, in a given C2 domain only a subset of the four sites may actually bind Ca 2+ in solution, since not all the sites provide adequate Ca 2+ coordination. Thus, the Ca 2+ -loaded C2 domains characterized to date possess from one to three Ca 2+ bound in different combinations of sites I-IV (12,15,(17)(18)(19).…”

“…Following docking, the separate catalytic domain hydrolytically releases arachidonic acid from lipid substrates, thereby triggering a variety of pathways including the inflammation response targeted by common anti-inflammatory drugs (22,23). The isolated cPLA 2 α C2 domain is activated by the binding of two Ca 2+ ions in solution with positive cooperativity, and this activation dramatically increases the affinity of the domain for membrane docking (18). High-resolution structures of the free domain reveal that the two bound Ca 2+ ions occupy sites I and II of the four potential sites (15,24), but it is not known which of the two sites are loaded first during the activation process, and much remains to be learned about the mechanism of positive cooperativity for C2 domains in general.…”