title: Binding of ricin A-chain to negatively charged phospholipid vesicles leads to protein structural changes and destabilizes the lipid bilayer.
abstract: Ricin is a heterodimeric protein toxin in which a catalytic polypeptide (the A-chain or RTA) is linked by a disulfide bond to a cell-binding polypeptide (the B-chain or RTB). During cell entry, ricin undergoes retrograde vesicular transport to reach the endoplasmic reticulum (ER) lumen, from where RTA translocates into the cytosol, probably by masquerading as a substrate for the ER-associated protein degradation (ERAD) pathway. In partitioning studies in Triton X-114 solution, RTA is predominantly found in the detergent phase, whereas ricin holotoxin, native RTB, and several single-chain ribosome-inactivating proteins (RIPs) are in the aqueous phase. Fluorescence spectroscopy and far-UV circular dichroism (CD) demonstrated significant structural changes in RTA as a result of its interaction with liposomes containing negatively charged phospholipid (POPG). These lipid-induced structural changes markedly increased the trypsin sensitivity of RTA and, on the basis of the protein fluorescence determinations, abolished its ability to bind to adenine, the product resulting from RTA-catalyzed depurination of 28S ribosomal RNA. RTA also released trapped calcein from POPG vesicles, indicating that it destabilized the lipid bilayer. We speculate that membrane-induced partial unfolding of RTA during cell entry may facilitate its recognition as an ERAD substrate.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Day
authors.forename: Teresa J T
authors.initials: TJ
authors.lastname: Pinheiro
authors.forename: Lynne M
authors.initials: LM
authors.lastname: Roberts
authors.forename: J Michael
authors.initials: JM
authors.lastname: Lord
mesh_terms: D005452:Fluoresceins / Q000378:metabolism; D008051:Lipid Bilayers; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D012276:Ricin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005452:Fluoresceins; D008051:Lipid Bilayers; D010743:Phospholipids; D012276:Ricin; C007740:fluorexon