Previous work has established that the 61 amino acid stretch from residue 322 to 382 in the T-domain of diphtheria toxin forms channels indistinguishable in ion-conducting properties from those formed by the entire T-domain. In the crystal structure of the toxin's water-soluble form, the bulk of this stretch is an α-helical hairpin, designated TH8-9. The present study was directed at determining which residues in TH8-9 line the ion-conducting pathway of the channel; i.e., its lumen or entrances. To this end, we singly mutated 49 of TH8-9's 51 residues (328–376) to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and then determined whether they reacted with small, charged, lipid-insoluble, sulfhydryl-specific methanethiosulfonate (MTS) derivatives added to the bathing solutions. The indication of a reaction, and that the residue lined the ion-conducting pathway, was a sudden change in single-channel conductance and/or flickering behavior. The results of this study were surprising in two respects. First, of the 49 cysteine-substituted residues in TH8-9 tested, 23 reacted with MTS derivatives in a most unusual pattern consisting of two segments: one extending from 329 to 341 (11 of 13 reacted), and the other from 347 to 359 (12 of 13 reacted); none of the residues outside of these two segments appeared to react. Second, in every cysteine mutant channel manifesting an MTS effect, only one transition in single-channel conductance (or flickering behavior) occurred, not the several expected for a multimeric channel. Our results are not consistent with an α-helical or β-strand model for the channel, but instead suggest an open, flexible structure. Moreover, contrary to common sense, they indicate that the channel is not multimeric but is formed from only one TH8-9 unit of the T-domain.
Probing the Structure of the Diphtheria Toxin Channel : Reactivity in Planar Lipid Bilayer Membranes of Cysteine-substituted Mutant Channels with Methanethiosulfonate Derivatives
Address correspondence to Alan Finkelstein, Department of Physiology & Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Ave. Bronx, New York 10461. FAX: 718-430-8819. E-mail: finkelst @aecom.yu.edu
It should be understood that calling these residues “MTS inaccessible” is an overinterpretation of the data. Such residues could have reacted with the reagent, but the addition of the −SCH2CH2R group to the cysteine might not have resulted in a change of single-channel conductance or flickering. We feel that for most of the residues, this is an unlikely possibility (see discussion).
The carboxy terminus of the T-domain is Pro 378. The construct in the work cited here terminated at Pro 382, thereby including four of the residues between the T- and the R-domain.
Abbreviations used in this paper: DTT, dithiothreitol; MTS, methanethiosulfonate.
H. Zhan's present address is Arris Pharmaceutical Corp., South San Francisco, CA 94080.
The issue of the lipid insolubility of these reagents, particularly the neutral form of MTS-EA, is addressed later.
There was, however, a major difference in the voltage gating behavior. The channels formed by all the mutant proteins retaining the histidine tag rapidly closed at small negative potentials (−10 to −60 mV), whereas those from which the histidine tag was removed closed very slowly at these voltages. This interesting phenomenon, which will be the subject of a later paper, is not relevant to the experiments described here.
The one exception was residue 363, which was tested with MTS-ES− added to the trans side and MTS-ET+ added to the cis side, under the pH conditions cis pH 7.0/trans pH 7.2.
This was generally true also for MTS-EA+. The few possible exceptions that we observed can be attributed to the membrane permeability of its neutral form (Holmgren et al., 1996), which, having traversed the membrane to the cis side, could now react with cysteine-modified mutant either in solution or at the membrane surface.
In the presumed heptameric channel formed by the PA63 component of anthrax toxin, several (not one!) step changes of conductance were produced by MTS-ET+ on the N306C mutant channel (unpublished data). Although not directly relevant to the present study of the diphtheria toxin channel, this does show that the substituted-cysteine-accessibility method employed here is capable of displaying multiple reactions in a multimeric channel.
Paul D. Huynh, Can Cui, Hangjun Zhan, Kyoung Joon Oh, R. John Collier, Alan Finkelstein; Probing the Structure of the Diphtheria Toxin Channel : Reactivity in Planar Lipid Bilayer Membranes of Cysteine-substituted Mutant Channels with Methanethiosulfonate Derivatives. J Gen Physiol 1 September 1997; 110 (3): 229–242. doi: https://doi.org/10.1085/jgp.110.3.229
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