Nat Struct Biol. C-terminal amino acid residues. The truncated proteins still formed sodium dodecyl sulfate-stable oligomers but were unable to bind to cells. Our data indicate that the C terminus of C2II mediates binding of the protein to cells and that the 7 C-terminal amino acids are structurally important for receptor binding. The actin-ADP-ribosylating C2 toxin from types C and D belongs to the family of toxins which consist of two separate proteins, an enzyme component, C2I, and a binding component, C2II (2, 7, 32). Further members of this toxin family are iota toxin from (27), ADP-ribosyltransferase (24), toxin (23), and the vegetative insecticidal proteins produced by (13). The C2I enzyme component of C2 toxin ADP-ribosylates G-actin at Arg-177 (1). ADP-ribosylation inhibits actin polymerization (1) and actin ATPase activity (12) and turns actin into a capping protein that 7-Methylguanosine binds to the barbed ends of actin filaments, inhibiting fast polymerization (30). Moreover, ADP-ribosylation of actin complexed with gelsolin alters the nucleation of the gelsolin-actin complex (32). In intact cells, C2 toxin causes redistribution of the actin cytoskeleton, depolymerization of actin filaments, and rounding up (25, 31, 33). Cellular uptake of C2I depends on the binding and translocation component C2II. C2II binds specifically to asparagine-linked complex carbohydrates, which act as toxin receptors on the surfaces of target cells (9). For efficient binding and translocation, C2II has to be activated by trypsin cleavage; thereby, an N-terminal 20-kDa fragment of C2II is released (20). Trypsin-activated C2II (59.8 kDa) oligomerizes to heptamers and forms channels in artificial membranes (3). After endocytosis of the C2II-C2I complex, translocation of the enzyme component into the cytosol occurs most likely from an acidic endosomal compartment (3). Recent cloning and sequencing of the gene encoding the binding component of C2 toxin revealed significant sequence similarities with the genes of binding components of the other actin-ADP-ribosylating toxins but also with the 7-Methylguanosine gene of the protective antigen (PA) of (15). PA is the binding component of the tripartite anthrax Rabbit polyclonal to PPP1R10 toxin (18) and translocates the edema factor, an adenylyl cyclase (17), and/or the lethal factor, a mitogen-activated protein kinase-cleaving metalloprotease (8), into the cytosol. Basing our work mainly on the crystal structure of PA (22), which is characterized by a four-domain structure, and its sequence similarity with the binding component of C2 toxin, we performed a structure-function analysis of C2II. Deduced from the primary sequence, C2II could be divided into four domains like those of PA. Whereas domains 1 to 3 (D1 to D3) of C2II show sequence similarities with those domains in PA, D4 is dissimilar to D4 in PA. Here we report that C-terminal D4 of C2II (C2II-D4), which covers amino acid residues 592 to 721, mediates cell surface binding of C2 toxin. Deletion analysis suggested that the 7 C-terminal amino acid residues of this domain are essential for cell binding. MATERIALS AND METHODS Materials. Oligonucleotides were obtained from MWG Biotech (Ebersberg, Germany). The pGEX-2T vector was included in the glutathione polymerase was purchased from Roche Molecular Diagnostics. Donkey anti-rabbit antibody coupled to horseradish peroxidase and an enhanced chemiluminescence detection kit were from Amersham (Braunschweig, Germany). The nitrocellulose-blotting membrane was from Schleicher and Schuell (Dassel, Germany). Glutathione-Sepharose 4B and protein ACSepharose-CL 4B were obtained from Pharmacia Biotech. Cell culture medium was purchased from Biochrom (Berlin, Germany), and fetal calf serum was obtained from PAN Systems (Aidenbach, Germany). Thrombin was obtained from Sigma (Deisenhofen, Germany). Trypsin and trypsin inhibitor were from Boehringer. Hanks’ balanced salt solution (HBSS) contained (concentrations in grams per liter in parentheses) CaCl2 (0.185), MgSO4 (0.089), KCl (0.4), KH2PO4 (0.06), NaCl (8.0), Na2HPO4 (0.048), and glucose (1.0), to which 10 mM HEPES 7-Methylguanosine (pH 7.4) was added. C2II of was purified as described previously (10). The N-terminal sequencing of trypsin-activated C2II was carried out by C. C. Shone Centre for Applied Microbiology and Research, Salisbury, United Kingdom). Cloning of the C2II gene. The C2II gene was amplified by PCR with 30 ng of partially KZZ1577(92-13) in a total volume of 100 l 7-Methylguanosine with 1 U of DNA polymerase in a reaction mixture that included deoxynucleoside triphosphates (100 M each) and 50 pmol of the primers C2II-pos (5-GATGGACCATGGCGGTTTCAAAATTTGAGAAC-3), which contains an DNA polymerase in a reaction mixture that included deoxynucleoside triphosphates (100 M each) and 50 pmol of the primers C2II-5 (5-GCTTCGGGATCCATGTTAGTTTCAAAATTTGAG-3), which contains a BL21 cells harboring the separate DNA fragments in plasmid pGEX-2T. Proteins were purified as described previously (4) and eluted with 10 mM glutathioneC100 mM NaClC50 mM Tris (pH 8.0) or incubated with thrombin (3.25 National Institutes of Health units/ml of bead suspension) for cleavage of the fusion proteins from GST. Thereafter, the suspension was centrifuged.