Solution structure and membrane interactions of the C2 domain of cytosolic phospholipase A2.
The amino-terminal, 138 amino acid C2 domain of cytosolic phospholipase A2 (cPLA2-C2) mediates an initial step in the production of lipid mediators of inflammation: the Ca2+-dependent translocation of the enzyme to intracellular membranes with subsequent liberation of arachidonic acid. The high resolution solution structure of this Ca2+-dependent, lipid-binding domain (CaLB) has been determined using heteronuclear three-dimensional NMR spectroscopy. Secondary structure analysis, derived from several sets of spectroscopic data, shows that the domain is composed of eight antiparallel beta-strands with six interconnecting loops that fits the "type II" topology for C2 domains. Using a total of 2370 distance and torsional restraints, the structure was found to be a beta-sandwich in the "Greek key" motif. The solution structure of cPLA2-C2 domain is very similar to the X-ray crystal structure of the C2 domain of phospholipase-C-delta and phylogenetic analysis clarifies the structural role of highly conserved residues. Calorimetric studies further demonstrate that cPLA2-C2 binds two Ca2+ with observed Kds of approximately 2 microM in an entropically assisted process. Moreover, regions on cPLA2-C2 interacting with membranes were identified by 15N-HSQC-spectroscopy of cPLA2-C2 in the presence of low molecular weight lipid micelles. An extended binding site was identified that binds the phosphocholine headgroup in a Ca2+-dependent manner and also interacts with proximal regions of the membrane surface. Based upon these results, a structural model is presented for the mechanism of association of cPLA2 with its membrane substrate.
Small Molecule Drug Discovery, Genetics Institute, 87 Cambridge Park Drive, Cambridge, MA 02140, USA.
G Y
GY
Xu
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T
McDonagh
H A
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Yu
E A
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Nalefski
J D
JD
Clark
D A
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Cumming
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D008823:Micelles; D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium