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 p roteins can assemble into lipid bilayers are relevant to several areas of biology, including the biosynthesis of membrane and secreted prote ins, virus membrane fusion and the action of immune proteins such as c omplement and perforin. The alpha-hemolysin (alpha HL) protein, an exo toxin secreted by Staphylococcus aureus that forms heptameric pores in lipid bilayers, is a useful model for studying membrane protein assem bly. In addition, modified alpha HL might be useful as a component of biosensors or in drug delivery. We have therefore used protein enginee ring to produce variants of alpha HL that contain molecular triggers a nd switches with which pore-forming activity can be modulated at will. Previously, we showed that the conductance of pores formed by the mut ant hemolysin alpha HL-H5, which contains a Zn(II)binding pentahistidi ne sequence, is blocked by Zn(II) from either side of the lipid bilaye r, 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 preven ting an impermeable oligomeric prepore from proceeding to the fully as sembled state. The prepore is a heptamer. Limited proteolysis shows th at, unlike the functional pore, the prepore contains sites near the am ino terminus of the polypeptide chain that are exposed to the aqueous phase. Upon removal of the bound Zn(II) with EDTA, pore formation is c ompleted and the sites near the amino terminus become occluded. Conver sion 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 int ermediate in assembly. Genetically-engineered switches for trapping an d releasing intermediates that are actuated by metal coordination or o ther chemistries might be generally useful for analyzing the assembly of membrane proteins and other supramolecular structures.