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Ca2+-signaling cycle of a membrane-docking C2 domain
E A Nalefski1, M M Slazas, J J Falke
1Department of Chemistry and Biochemistry, University of Colorado at Boulder 80309-0215, USA.
Biochemistry
|October 27, 1997
Summary
The C2 domain in human cytosolic phospholipase A2 (cPLA2) binds two calcium ions cooperatively, enabling membrane docking. This binding is slower and sequential when the protein is membrane-bound, crucial for initiating inflammatory responses.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- The C2 domain is a calcium-dependent, membrane-targeting motif found in many signal-transducing proteins.
- Cytosolic phospholipase A2 (cPLA2) utilizes its C2 domain to initiate the inflammatory response by hydrolyzing lipids upon calcium signaling.
Purpose of the Study:
- To investigate the calcium-binding and dissociation kinetics of the human cPLA2 C2 domain.
- To elucidate the mechanism of membrane recruitment and release mediated by the C2 domain.
Main Methods:
- Equilibrium binding experiments
- Stopped-flow kinetic studies
- Analysis of protein-membrane interactions
Main Results:
- The human cPLA2 C2 domain binds two calcium ions with positive cooperativity, inducing a conformational change and membrane docking.
- Calcium dissociation from the isolated domain is rapid and simultaneous, but becomes slow and sequential when membrane-bound due to occluded binding sites.
- The C2 domain remains membrane-bound until the second calcium ion dissociates, highlighting a critical step in the signaling cycle.
Conclusions:
- The calcium-signaling cycle of the cPLA2 C2 domain involves a stable, membrane-bound state with two occluded calcium ions.
- One calcium ion is essential for maintaining the protein-membrane complex, underscoring its role in regulating inflammatory pathways.