STRUCTURE-FUNCTION-RELATIONSHIPS IN DIPHTHERIA-TOXIN CHANNELS .1. 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 inactiva ting their protein synthetic machinery; its C-terminal B chain is requ ired for the binding of toxin to sensitive cells and for the transloca tion of the A fragment into the cytosol. This B fragment, consisting o f its N-terminal T domain (amino acids 191-386) and its C-terminal R d omain (amino acids 387-535) is responsible for the ion-conducting chan nels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channel s formed by deletion mutants of DT in lipid bilayer membranes under se veral 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 (I1- 2), have single channel conductances and selectivities essentially ide ntical to those of channels formed by wild-type DT. Furthermore, delet ing the N-terminal 118 amino acids of the T domain also has minimal ef fect on the single channel conductance and selectivity of the mutant c hannels. 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.