Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins.
J Gen Physiol
; 137(4): 343-56, 2011 Apr.
Article
in En
| MEDLINE
| ID: mdl-21402886
ABSTRACT
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 â¼100 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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Bacterial Toxins
/
Antigens, Bacterial
Type of study:
Prognostic_studies
Language:
En
Journal:
J Gen Physiol
Year:
2011
Document type:
Article
Affiliation country:
United States