The diptheria toxin channel in planar lipid bilayers: Studies on its pH -dependent formation and identification of the residues lining its ion -conduction pathway

Abstract

Diphtheria Toxin (DT) is a 535-amino acid polypeptide secreted by Corynebacterium diphtheria. Upon mild proteolysis and reduction, the toxin separates into two fragments: the N-terminal A fragment, that kills cells by enzymatically inhibiting their protein synthetic machinery, and the C-terminal B fragment, that is required for the binding of the toxin to the cell surface and for translocation of the A fragment from the extracellular space to the cytosol. The machinery for this latter process resides in the B fragment's N-terminal 172 residues, known as the T-domain, which forms ion-conducting channels in planar lipid bilayers at acidic pH. The T-domain consists of ten alpha-helices (designated THI-TH9 and TH5'). It has been shown that the 61-amino acid segment from residue 322 to 382 in the T-domain is all that is required to form the ion-conducting channel. In the crystal structure of the water-soluble form of the toxin, most of this 61-amino acid stretch consists of an alpha-helical hairpin, designated TH8-9, which contains helices TH8 (19 amino acids), TH9 (21 amino acids), and the interhelical loop (9 amino acids) connecting them.;This thesis reports my studies, in planar lipid bilayers, of the pH dependence of DT channel formation and of the identification of the residues that line the channel's ion-conducting pathway. Chapter 1 gives a general introduction to what is known about the action of diphtheria toxin, its channel-forming properties, and its water-soluble structure. Chapters 2 and 3 deal with the pH dependence of channel formation. The former shows that linking, via disulfide bonds, the interhelical loop of TH8-9 to loops outside of this region inhibits the low pH-induced exposure of TH8-9 and dramatically inhibits channel-forming ability. The latter chapter identifies the two acidic residues in the interhelical loop of TH8-9, Glu 349 and Asp 352, as responsible for most of the trans pH dependence of channel formation. Chapters 4 and 5, which are the main focus of this thesis, deal with the identification of residues lining the ion-conducting pathway of the channel. Cysteines were substituted, one at a time, at 49 continuous positions (residues 328--376) in TH8-9, and their accessibility to small, charged, lipid-insoluble, methanethiosulfonate (MTS) derivatives was determined. Chapter 4 describes the phenomenology of the single-channel response to these MITS-derivatives at four sites in the TH8-9 interhelical loop, and Chapter 5 extends these findings to the rest of TH8-9. The results obtained in Chapter 5 reveal a very unusual distribution for the residues lining the ion-conducting pathway, that is inconsistent with either an alpha-helical or beta-strand structure for the channel. In addition, the nature of the response of cysteine-mutant channels to the MTS derivatives surprisingly leads to the suggestion that the diphtheria toxin channel is not multimeric, but is instead formed from one TH8-9 unit. The last chapter (Chapter 6) summarizes the findings of the preceding 4 chapters and discusses future research directions.