Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain.
Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C-terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191-386) and its C-terminal R domain (amino acids 387-535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (IL-2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes alpha-helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.
Silverman
J A
JA
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Mindell
J A
JA
Zhan
H
H
Finkelstein
A
A
Collier
R J
RJ
D000595:Amino Acid Sequence; D004167:Diphtheria Toxin / Q000032:analysis* / Q000378:metabolism* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007376:Interleukin-2 / Q000494:pharmacology; D007473:Ion Channels / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D008051:Lipid Bilayers / Q000737:chemistry; D008564:Membrane Potentials / Q000502:physiology; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009154:Mutation; D013329:Structure-Activity Relationship
D004167:Diphtheria Toxin; D007376:Interleukin-2; D007473:Ion Channels; D008051:Lipid Bilayers; D008567:Membranes, Artificial