Clostridium botulinum C2 toxin. Identification of the binding site for chloroquine and related compounds and influence of the binding site on properties of the C2II channel.
Clostridium botulinum C2 toxin belongs to the family of binary AB type toxins that are structurally organized into distinct enzyme (A, C2I) and binding (B, C2II) components. The proteolytically activated 60-kDa C2II binding component is essential for C2I transport into target cells. It oligomerizes into heptamers and forms channels in lipid bilayer membranes. The C2II channel is cation-selective and can be blocked by chloroquine and related compounds. Residues 303-330 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which has been implicated in the formation of two amphipathic beta-strands involved in membrane insertion and channel formation. In the present study, C2II mutants created by substitution of different negatively charged amino acids by alanine-scanning mutagenesis were analyzed in artificial lipid bilayer membranes. The results suggested that most of the C2II mutants formed SDS-resistant oligomers (heptamers) similar to wild type. The mutated negatively charged amino acids did not influence channel properties with the exception of Glu(399) and Asp(426), which are probably localized in the vestibule near the channel entrance. These mutants show a dramatic decrease in their affinity for binding of chloroquine and its analogues. Similarly, F428A, which represents the Phi-clamp in anthrax protective antigen, was mutated in C2II in several other amino acids. The C2II mutants F428A, F428D, F428Y, and F428W not only showed altered chloroquine binding but also had drastically changed single channel properties. The results suggest that amino acids Glu(399), Asp(426), and Phe(428) have a major impact on the function of C2II as a binding protein for C2I delivery into target cells.
Neumeyer
Tobias
T
Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
Schiffler
Bettina
B
Maier
Elke
E
Lang
Alexander E
AE
Aktories
Klaus
K
Benz
Roland
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers / Q000378:metabolism; D001905:Botulinum Toxins / Q000737:chemistry / Q000378:metabolism*; D002738:Chloroquine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007473:Ion Channels / Q000378:metabolism*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D001704:Biopolymers; D007473:Ion Channels; D002738:Chloroquine; D001905:Botulinum Toxins; C096324:botulinum toxin type C