title: Characterization of the Lipid-Binding Site of Equinatoxin II by NMR and Molecular Dynamics Simulation.
abstract: Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-(15)N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation.
authors.affiliation: School of Chemistry, University of Melbourne, Victoria, Australia; Bio21 Institute, University of Melbourne, Victoria, Australia.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Weber
authors.affiliation: Bio21 Institute, University of Melbourne, Victoria, Australia.
authors.forename: Shenggen
authors.initials: S
authors.lastname: Yao
authors.affiliation: Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
authors.forename: Nejc
authors.initials: N
authors.lastname: Rojko
authors.affiliation: Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.affiliation: Center for Structural Biology, Department of Chemistry, Vanderbilt University, Nashville, Tennessee.
authors.forename: Terry P
authors.initials: TP
authors.lastname: Lybrand
authors.affiliation: IBM Research Collaboratory for Life Sciences, Victorian Life Sciences Computation Initiative, University of Melbourne, Victoria, Australia.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Downton
authors.affiliation: IBM Research Collaboratory for Life Sciences, Victorian Life Sciences Computation Initiative, University of Melbourne, Victoria, Australia.
authors.forename: John
authors.initials: J
authors.lastname: Wagner
authors.affiliation: School of Chemistry, University of Melbourne, Victoria, Australia; Bio21 Institute, University of Melbourne, Victoria, Australia. Electronic address: fs@unimelb.edu.au.
authors.forename: Frances
authors.initials: F
authors.lastname: Separovic
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D013109:Sphingomyelins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003064:Cnidarian Venoms; D013109:Sphingomyelins; D010767:Phosphorylcholine; C028810:dodecylphosphocholine; C012307:equinatoxin