Interactions of the GM2 activator protein with phosphatidylcholine bilayers: a site-directed spin-labeling power saturation study.
The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.
Mathias
Jordan D
JD
Department of Chemistry, University of Florida, Gainesville, Florida, USA.
Ran
Yong
Y
Carter
Jeffery D
JD
Fanucci
Gail E
GE
D002614:Chelating Agents; D004578:Electron Spin Resonance Spectroscopy; D049289:G(M2) Activator Protein / Q000737:chemistry* / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D009532:Nickel; D010713:Phosphatidylcholines / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism; D013113:Spin Labels; D014867:Water / Q000737:chemistry
D002614:Chelating Agents; D049289:G(M2) Activator Protein; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D013113:Spin Labels; D014867:Water; D009532:Nickel; C028694:1-palmitoyl-2-oleoylphosphatidylcholine