title: Unusual mode of binding of human group IIA secreted phospholipase A2 to anionic interfaces as studied by continuous wave and time domain electron paramagnetic resonance spectroscopy.
abstract: Human group IIA phospholipase A(2) (hGIIA) is secreted from a number of cells during inflammation and is known to interact strongly with anionic membranes and to exhibit potent Gram-positive bactericidal activity. This protein contains 23 cationic residues, which are scattered over its entire surface, resulting in a high pI of 9.39. To understand the molecular basis for the selective binding of hGIIA to anionic membranes, 14 single-site, spin-labeled hGIIA proteins were analyzed in the presence and absence of vesicles of anionic phospholipid by time domain and continuous wave electron paramagnetic resonance (EPR) spin relaxant techniques. Surprisingly, for hGIIA bound to anionic vesicles, all of the spin labels were highly protected from water-soluble spin relaxants. Together with light scattering studies, these EPR results suggest the formation of a supramolecular aggregate involving clusters of hGIIA molecules bridging together multiple vesicles. This anomalous mode of binding of hGIIA to anionic phospholipid explains previous data in which charge reversal mutation of a few cationic residues on multiple faces of hGIIA leads to a comparable and modest reduction in affinity of the protein for anionic vesicles. In the presence of mixed micelles composed of 10% anionic phospholipids in Triton X-100 a monodisperse protein-lipid complex is formed. Under these conditions, the EPR methods were used to map the surface of hGIIA that constitutes the interfacial binding site (IBS). The IBS of hGIIA consists of the highly hydrophobic surface that surrounds the opening to the active site slot.
authors.affiliation: Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Canaan
authors.forename: Robert
authors.initials: R
authors.lastname: Nielsen
authors.forename: Farideh
authors.initials: F
authors.lastname: Ghomashchi
authors.forename: Bruce H
authors.initials: BH
authors.lastname: Robinson
authors.forename: Michael H
authors.initials: MH
authors.lastname: Gelb
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*
chemical_list: D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2