The topology and molecularity of the channel formed by diptheria toxin's T domain or fear and loathing in da Bronx
Gordon, Michael Brian
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Diphthera toxin (DT), is a 535 amino acid peptide secreted by Corynebacterium diphtheriae. This protein is composed of three functional domains: (i) the A fragment, a catalytic domain that kills cells by inhibiting protein synthesis, (ii) the T domain, a transmembrane fragment that forms an ion channel, and (iii) a receptor-binding domain that localizes the toxin to specific mammalian cells. We have previously shown that the T domain is capable of translocating the entire catalytic domain across model, planar phospholipid bilayers in the absence of other proteins. During this translocation, the T domain forms a cation-selective channel of approximately 40 pS in 1M KCl with a pH gradient of 5.3 to 7.2 across the bilayer.;We have developed a model for the topology of the T domain as seen in the open channel state (the state of the channel when the catalytic domain has been translocated). This model, which contains three membrane-spanning segments, was derived from experiments in which either a histidine tag (H6 tag) or biotin was attached at residues that were mutated to cysteines. The H6 tag, used to purify the T domain on a Ni+2 column, was shown to block the channel conductance in a voltage dependent manner.;Mixtures were made of two T domain constructs that have distinguishable voltage-dependent blocking characteristics. One of these constructs contained an N-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a C-terminal H6 tag that blocks the channel at positive voltages. If the channel were constructed from multiple T domain subunits, one would expect to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. We therefore conclude that the T domain channel is monomeric. (Abstract shortened by UMI.).