An intermediate in the assembly of a pore-forming protein trapped with a genetically-engineered switch.
BACKGROUND
Studies of the mechanisms by which certain water-soluble proteins can assemble into lipid bilayers are relevant to several areas of biology, including the biosynthesis of membrane and secreted proteins, virus membrane fusion and the action of immune proteins such as complement and perforin. The alpha-hemolysin (alpha HL) protein, an exotoxin secreted by Staphylococcus aureus that forms heptameric pores in lipid bilayers, is a useful model for studying membrane protein assembly. In addition, modified alpha HL might be useful as a component of biosensors or in drug delivery. We have therefore used protein engineering to produce variants of alpha HL that contain molecular triggers and switches with which pore-forming activity can be modulated at will. Previously, we showed that the conductance of pores formed by the mutant hemolysin alpha HL-H5, which contains a Zn(II)-binding pentahistidine sequence, is blocked by Zn(II) from either side of the lipid bilayer, suggesting that residues from the pentahistidine sequence line the lumen of the transmembrane channel.


RESULTS
Here we show that Zn(II) can arrest the assembly of alpha HL-H5 before pore formation by preventing an impermeable oligomeric prepore from proceeding to the fully assembled state. The prepore is a heptamer. Limited proteolysis shows that, unlike the functional pore, the prepore contains sites near the amino terminus of the polypeptide chain that are exposed to the aqueous phase. Upon removal of the bound Zn(II) with EDTA, pore formation is completed and the sites near the amino terminus become occluded. Conversion of the prepore to the active pore is the rate-determining step in assembly and cannot be reversed by the subsequent addition of excess Zn(II).


CONCLUSIONS
The introduction of a simple Zn(II)-binding motif into a pore-forming protein has allowed the isolation of a defined intermediate in assembly. Genetically-engineered switches for trapping and releasing intermediates that are actuated by metal coordination or other chemistries might be generally useful for analyzing the assembly of membrane proteins and other supramolecular structures.
Walker
B
B
Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545-2737, USA.
Braha
O
O
Cheley
S
S
Bayley
H
H
D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry*; D001665:Binding Sites; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000201:enzymology; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008051:Lipid Bilayers; D008956:Models, Chemical; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D015032:Zinc / Q000378:metabolism
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; C021091:staphylococcal alpha-toxin; D004492:Edetic Acid; D019286:Endopeptidase K; D015032:Zinc