The CLC proteins form a broad family of anion-selective transport proteins that includes both channels and exchangers. Despite extensive structural, functional, and computational studies, the transport mechanism of the CLC exchangers remains poorly understood. Several transport models have been proposed but have failed to capture all the key features of these transporters. Multiple CLC crystal structures have suggested that a conserved glutamic acid, Glu ex , can adopt three conformations and that the interconversion of its side chain between these states underlies H + /Cl − exchange. One of these states, in which Glu ex occupies the central binding site (S cen ) while Cl − ions fill the internal and external sites (S int and S ext ), has only been observed in one homologue, the eukaryotic cmCLC. The existence of such a state in other CLCs has not been demonstrated. In this study, we find that during transport, the prototypical prokaryotic CLC exchanger, CLC-ec1, adopts a conformation with functional characteristics that match those predicted for a cmCLC-like state, with Glu ex trapped in S cen between two Cl − ions. Transport by CLC-ec1 is reduced when [Cl − ] is symmetrically increased on both sides of the membrane and mutations that disrupt the hydrogen bonds stabilizing Glu ex in S cen destabilize this trapped state. Furthermore, inhibition of transport by high [Cl − ] is abolished in the E148A mutant, in which the Glu ex side chain is removed. Collectively, our results suggest that, during the CLC transport cycle, Glu ex can occupy S cen as well as the S ext position in which it has been captured crystallographically and that hydrogen bonds with the side chains of residues that coordinate ion binding to S cen play a role in determining the equilibrium between these two conformations.

Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA 63 ) 7 , formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell’s endosomal membrane, disrupting cellular homeostasis. (PA 63 ) 7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF N , the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF N molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF N s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF N bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF N bound. We also present a simple electrodiffusion model of translocation in which LF N is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA 63 ) 7 , in the host cell’s endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA 63 ) 7 incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ∼100 Å–long, 15 Å–wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LF N (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LF N adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LF N in the normal direction but also can, at least partially, translocate LF N in the opposite direction.

The toxin produced by Bacillus anthracis , the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA 63 ) 7 , formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA 63 ) 7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LF N (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA 63 ) 7 channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LF N enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton–protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO 3 − , which is essentially not titratable, is introduced at most positions in LF N , through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF N translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA 63 ) 7 channel resides at or near its Φ-clamp, the ring of seven phenylalanines near the channel's entrance.