Role of lysine residues in membrane anchoring of saposin C.
Molecular dynamics (MD) simulations of the N-terminal region of saposin C, containing amino acid residues 4-20 (saposin C4-20), were performed over 2.5 ns in 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) monolayers. The simulations revealed several strong specific interactions of lysine 13 (Lys13) and lysine 17 (Lys17) in saposin C4-20 with the anionic phospholipids, which are required for membrane anchoring of the peptide. Membrane anchoring of saposin C4-20 facilitates saposin C-induced liposomal membrane fusion. Substitutions of Lys13 or Lys17 with alanine or glutamic acid led to a substantial loss of saposin C's fusogenicity. However, arginine replacement of Lys13 or Lys17 caused a partial loss of saposin C's fusogenic activity. The membrane anchoring of saposin C was altered in the presence of 0.4 M sodium chloride. Differential salt effects on Lys-mutant saposin Cs were observed using Trp fluorescence analysis. Low salt concentration had a more significant impact on Lys-mutant saposin C with a negatively charged amino acid residue replacement than those mutants with a positively charged or neutral residue replacement. These results indicate that positively charged amino acids at positions 13 and 17 are required for the fusogenic function of saposin C. In addition, the side-chain structure of lysine is crucial to the precise membrane anchoring which is necessary for the total fusion activity of saposin C. The MD simulations and vesicle size measurements of lysine-mutant saposins confirm the importance of the two lysine residues in saposin C4-20 for saposin C-induced fusion of negatively charged phospholipid membranes.
Liu
Anping
A
Department of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, OH 45221-0172, USA.
Wenzel
Nathan
N
Qi
Xiaoyang
X
D001665:Binding Sites; D003198:Computer Simulation; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D008239:Lysine / Q000737:chemistry*; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D049231:Saposins / Q000737:chemistry*; D013329:Structure-Activity Relationship
D017261:Glycosylphosphatidylinositols; D008563:Membrane Lipids; D008565:Membrane Proteins; D049231:Saposins; D008239:Lysine