Abstract
Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.
Highlights
The conserved EF-hand (EF) domain is necessary for active phospholipase
No calcium ion is found associated with the EF-hand domain that is visible in crystal structures of phospholipase C (PLC) ␦1, even though the enzyme is soaked in calcium or its analogs [7, 25,26,27]
PIP2 Binding Cannot Substitute for the Loss in Relative Activity of EF-domain Mutants—Because the total lipid concentration in the vesicles used in the activity assays (10 or 5 mM) were well above the KD value for rEF or Phospholipase C ␦1 (PLC ␦1) partitioning onto binary component vesicles (0.14 to 0.25 mM for rEF binding to SUVs with a 1:1 anionic phospholipid to PC and 0.05 to 0.15 for PLCd1 binding to LUVs with a 1:1 anionic phospholipid), it is reasonable to assume the full-length PLC to be completely partitioned onto vesicles
Summary
The conserved EF-hand (EF) domain is necessary for active phospholipase. Results: EF binds to anionic phospholipid-containing vesicles; EF mutations introduced into PLC ␦1 reduce activity not recoverable with added PIP2. Recombinant EF-hand domain of phospholipase C ␦1 has a moderate affinity for anionic phospholipids in the absence of Ca2؉ that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence This region of PLC ␦1 is missing in the crystal structure. All members of the PLC family exhibit a multidomain structure that comprises the catalytic X and Y domains, the protein kinase C conserved region 2 (C2) domain, EF-hand domain, and pleckstrin homology (PH) domain (except for the sperm-specific PLC, which does not have a PH domain). Mutagenesis of specific cationic and hydrophobic residues in the separate EF-hand domain has modest effects on vesicle binding but significant effects on the activity of full-length PLC ␦1. Conserved residues in other PLC EFhand domains suggest this may be the primary function of this structural unit in all mammalian PLC enzymes
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