Barbara Walker

Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins.

Abstract Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin are well-studied prototypes of pore-forming bacterial cytotoxins. Each is produced as a water-soluble single-chain polypeptide that inserts into target membranes to form aqueous transmembrane pores. This review will compare properties of the three toxin prototypes, highlighting the similarities and also the differences in their structure, mode of binding, mechanism of pore formation, and the responses they elicit in target cells. Pore-forming toxins represent the most potent and versatile weapons with which invading microbes damage the host macroorganism.

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.

A photogenerated pore-forming protein

BACKGROUND The permeabilization of cells with bacterial pore-forming proteins is an important technique in cell biology that allows the exchange of small reagents into the cytoplasm of a cell. Another notable technology is the use of caged molecules whose activities are blocked by addition of photoremovable protecting groups. This allows the photogeneration of reagents on or in cells with spatial and temporal control. Here, we combine these approaches to produce a caged pore-forming protein for the controlled permeabilization of cells. RESULTS 2-Bromo-2-(2-nitrophenyl)acetic acid (BNPA), a water-soluble cysteine-directed reagent for caging peptides and proteins with the alpha-carboxy-2-nitrobenzyl (CNB) protecting group, was synthesized. Glutathione (gamma-Glu-Cys-Gly) was released in high yield from gamma-Glu-CysCNB-Gly by irradiation at 300 nm. Based on this finding, scanning mutagenesis was used to find a single-cysteine mutant of the pore-forming protein staphylococcal alpha-hemolysin (alpha HL) suitable for caging. When alpha HL-R104C was derivatized with BNPA, pore-forming activity toward rabbit erythrocytes was lost. Near UV irradiation led to regeneration of the cysteine sulfhydryl group and the restoration of pore-forming activity. CONCLUSIONS Caged pore-forming proteins are potentially useful for permeabilizing one cell in a collection of cells or one region of the plasma membrane of a single cell. Therefore, alpha HL-R104C-CNB and other caged proteins designed to create pores of various diameters should be useful for many purposes. For example, the ability to introduce reagents into one cell of a network or into one region of a single cell could be used in studies of neuronal modulation. Further, BNPA should be generally useful for caging cysteine-containing peptides and single-cysteine mutant proteins to study, for example, cell signaling or structural changes in proteins.

Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal α-hemolysin

Structural changes in staphylococcal α‐hemolysin (αHL) that occur during oligomerization and pore formation on membranes have been examined by using a simple gel‐shift assay to determine the rate of modification of key single‐cysteine mutants with the hydrophilic sulfhydryl reagent, 4‐acetamido‐4′‐((iodoacetyl)amino)stilbene‐2,2′‐disulfonate (IASD). The central glycine‐rich loop of αHL lines the lumen of the transmembrane channel. A residue in the loop remains accessible to IASD after assembly, in keeping with the ability of the pore to pass molecules of 1̃000 Da. By contrast, residues near the N‐terminus, which are critical for pore function, become deeply buried during oligomerization, while a residue at the extreme C‐terminus increases in reactivity after assembly, consistent with a location in the part of the pore that projects from the surface of the lipid bilayer.

Genetically Engineered Pores as Metal Ion Biosensors

We are adapting proteins that form pores in lipid bilayers for use as components of biosensors. Specifically, we have produced genetically engineered variants of the α hemolysin (αHL) from Staphylococcus aureus with properties that are sensitive to low concentrations of divalent cations. For example, the pore-forming activity of one mutant (αHL-H5: residues 130–134 inclusive replaced with histidine) is inhibited by Zn 2+ at concentrations as low as 1 μM, as judged by the reduction in its ability to lyse rabbit red blood cells and to increase the conductance of planar lipid bilayer membranes. When αHL-H5 is added to the aqueous phase bathing one side of a planar membrane, the subsequent addition of 100 μM Zn 2+ to either side blocks the pores that form. This result suggests that at least part of the mutated region lines the channel lumen. Ca 2+ and Mg 2+ do not block the channel and therefore the H5 mutation confers a degree of analyte specificity to the αHL pore. The results suggest that genetically engineered pores have great promise for the rapid and sensitive detection of metal cations and we discuss the merits and potential limitations for their use in this application. Specifically, we examine the issues of selectivity, sensitivity, response time, dynamic range and longevity. Some of these properties are interdependent. For example, the goals of high sensitivity and rapid response time can be in conflict.

Genetically Engineered Protein Pores as Components of Synthetic Microstructures

Proteinaceous, nanometer-scale pores might be used as components of new materials.1 For example, such pores could be incorporated into thin films to confer novel permeability properties upon them. These films in turn would have several technological applications, which are discussed below, including acting as molecular filters in sensors or as components of microelectronic devices. The development of genetically engineered pores for this purpose is timely as it can be based on three recent advances in molecular biology: the use of recombinant DNA technology to alter the structures of polypeptides at will;2,3 a rapidly increasing body of knowledge about the mechanisms of channel and pore proteins;4–6 and progress on the formation and structural analysis of 2D protein lattices.7,8 With this in mind, we have chosen to explore the properties of the α-hemolysin (αHL) from Staphylococcus aureus.9 This surprisingly hydrophilic 293 amino acid polypeptide10 is secreted by Staphylococcus as a monomer and assembles into a hexameric pore in target membranes such as those of rabbit red blood cells. Single channel recordings indicate that the pore is 1.1–1.2 nm in internal diameter.11 The monomer and the pore have similar secondary structures predominantly comprising β-sheet.12,13