The single C-terminal helix of human phospholipid scramblase 1 is required for membrane insertion and scrambling activity

摘要

Human phospholipid scramblase 1 (hPLSCRl) belongs to the ATP-inde-pendent class of phospholipid translocators which possess a single EF-hand-like Ca2 +-binding motif and also a C-terminal helix (CTH). The CTH domain of hPLSCRl was believed to be a putative single transmembrane helix at the C-terminus. Recent homology modeling studies by Bat-eman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A & Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCRl, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membraneanchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro recon-stitution studies confirm that the predicted CTH is required for membrane insertion and scramblingactivity. CTH deletion caused a 50% decrease in binding affinity of Ca~(2+) for ACTH-hPLSCRl (K_d = 115 mu m) compared with hPLSCRl (K_A = 249 mu m). Far UV-CD studies revealed that the CTH peptide adopts alpha-helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide-quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membranedepends on the presence of charge interactions. TOX-CAT assay revealed that CTH of hPLSCRl does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca~(2+) coordination and also plays an important role in the functional conformation of hPLSCRl.