Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient ß-barrel pore formation.

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging ß-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane ß-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane ß-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric ß-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional ß-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.

Elucidating liquid crystal-aqueous interface for the study of cholesterol-mediated action of a ß-barrel pore forming toxin.

Pore-forming toxins (PFTs) produced by pathogenic bacteria serve as prominent virulence factors with potent cell-killing activity. Most of the ß-barrel PFTs form transmembrane oligomeric pores in the membrane lipid bilayer in the presence of cholesterol. The pore-formation mechanisms of the PFTs highlight well-orchestrated regulated events in the membrane environment, which involve dramatic changes in the protein structure and organization. Also, concerted crosstalk between protein and membrane lipid components appears to play crucial roles in the process. Membrane-damaging lesions formed by the pore assembly of the PFTs would also be expected to impose drastic alterations in the membrane organization, details of which remain obscure in most of the cases. Prior reports have established that aqueous interfaces of liquid crystals (LCs) offer promise as responsive interfaces for biomolecular events (at physiologically relevant concentrations), which can be visualized as optical signals. Inspired by this, herein, we sought to understand the lipid membrane interactions of a ß-barrel PFT i.e., Vibrio cholerae cytolysin (VCC), using LC-aqueous interfaces. Our results show the formation of dendritic patterns upon the addition of VCC to the lipid embedded with cholesterol over the LC film. In contrast, we did not observe any LC reorientation upon the addition of VCC to the lipid-laden LC-aqueous interface in the absence of cholesterol. An array of techniques such as polarizing optical microscopy (POM), atomic force microscopy (AFM), and fluorescence measurements were utilized to decipher the LC response to the lipid interactions of VCC occurring at these interfaces. Altogether, the results obtained from our study provide a novel platform to explore the mechanistic aspects of the protein-membrane interactions, in the process of membrane pore-formation by the membrane-damaging PFTs.

Cryo-EM elucidates mechanism of action of bacterial pore-forming toxins.

Pore-forming toxins (PFTs) rupture plasma membranes and kill target cells. PFTs are secreted as soluble monomers that undergo drastic structural rearrangements upon interacting with the target membrane and generate transmembrane oligomeric pores. A detailed understanding of the molecular mechanisms of the pore-formation process remains unclear due to limited structural insights regarding the transmembrane oligomeric pore states of the PFTs. However, recent advances in the field of cryo-electron microscopy (cryo-EM) have led to the high-resolution structure determination of the oligomeric pore forms of diverse PFTs. Here, we discuss the pore-forming mechanisms of various PFTs, specifically the mechanistic details contributed by the cryo-EM-based structural studies.

Structures and functions of the membrane-damaging pore-forming proteins.

Pore-forming proteins (PFPs) of the diverse life forms have emerged as the potent cell-killing entities owing to their specialized membrane-damaging properties. PFPs have the unique ability to perforate the plasma membranes of their target cells, and they exert this functionality by creating oligomeric pores in the membrane lipid bilayer. Pathogenic bacteria employ PFPs as toxins to execute their virulence mechanisms, whereas in the higher vertebrates PFPs are deployed as the part of the immune system and to generate inflammatory responses. PFPs are the unique dimorphic proteins that are generally synthesized as water-soluble molecules, and transform into membrane-inserted oligomeric pore assemblies upon interacting with the target membranes. In spite of sharing very little sequence similarity, PFPs from diverse organisms display incredible structural similarity. Yet, at the same time, structure-function mechanisms of the PFPs document remarkable versatility. Such notions establish PFPs as the fascinating model system to explore variety of unsolved issues pertaining to the structure-function paradigm of the proteins that interact and act in the membrane environment. In this article, we discuss our current understanding regarding the structural basis of the pore-forming functions of the diverse class of PFPs. We attempt to highlight the similarities and differences in their structures, membrane pore-formation mechanisms, and their implications for the various biological processes, ranging from the bacterial virulence mechanisms to the inflammatory immune response generation in the higher animals.

Single-particle cryo-EM reveals conformational variability of the oligomeric VCC ß-barrel pore in a lipid bilayer.

Vibrio cholerae cytolysin (VCC) is a water-soluble, membrane-damaging, pore-forming toxin (PFT) secreted by pathogenic V. cholerae, which causes eukaryotic cell death by altering the plasma membrane permeability. VCC self-assembles on the cell surface and undergoes a dramatic conformational change from prepore to heptameric pore structure. Over the past few years, several high-resolution structures of detergent-solubilized PFTs have been characterized. However, high-resolution structural characterization of small ß-PFTs in a lipid environment is still rare. Therefore, we used single-particle cryo-EM to characterize the structure of the VCC oligomer in large unilamellar vesicles, which is the first atomic-resolution cryo-EM structure of VCC. From our study, we were able to provide the first documented visualization of the rim domain amino acid residues of VCC interacting with lipid membrane. Furthermore, cryo-EM characterization of lipid bilayer-embedded VCC suggests interesting conformational variabilities, especially in the transmembrane channel, which could have a potential impact on the pore architecture and assist us in understanding the pore formation mechanism.

Tyrosine in the hinge region of the pore-forming motif regulates oligomeric ß-barrel pore formation by Vibrio cholerae cytolysin.

ß-barrel pore-forming toxins perforate cell membranes by forming oligomeric ß-barrel pores. The most crucial step is the membrane-insertion of the pore-forming motifs that create the transmembrane ß-barrel scaffold. Molecular mechanism that regulates structural reorganization of these pore-forming motifs during ß-barrel pore-formation still remains elusive. Using Vibrio cholerae cytolysin as an archetypical example of the ß-barrel pore-forming toxin, we show that a key tyrosine residue (Y321) in the hinge region of the pore-forming motif plays crucial role in this process. Mutation of Y321 abrogates oligomerization of the membrane-bound toxin protomers, and blocks subsequent steps of pore-formation. Our study suggests that the presence of Y321 in the hinge region of the pore-forming motif is crucial for the toxin molecule to sense membrane-binding, and to trigger essential structural rearrangements required for the subsequent oligomerization and pore-formation process. Such a regulatory mechanism of pore-formation by V. cholerae cytolysin has not been documented earlier in the structurally related ß-barrel pore-forming toxins.

Sequence Diversity in the Pore-Forming Motifs of the Membrane-Damaging Protein Toxins.

Pore-forming proteins/toxins (PFPs/PFTs) are the distinct class of membrane-damaging proteins. They act by forming oligomeric pores in the plasma membranes. PFTs and PFPs from diverse organisms share a common mechanism of action, in which the designated pore-forming motifs of the membrane-bound protein molecules insert into the membrane lipid bilayer to create the water-filled pores. One common characteristic of these pore-forming motifs is that they are amphipathic in nature. In general, the hydrophobic sidechains of the pore-forming motifs face toward the hydrophobic core of the membranes, while the hydrophilic residues create the lining of the water-filled pore lumen. Interestingly, pore-forming motifs of the distinct subclass of PFPs/PFTs share very little sequence similarity with each other. Therefore, the common guiding principle that governs the sequence-to-structure paradigm in the mechanism of action of these PFPs/PFTs still remains an enigma. In this article, we discuss this notion using the examples of diverse groups of membrane-damaging PFPs/PFTs.

Taking Toll on Membranes: Curious Cases of Bacterial ß-Barrel Pore-Forming Toxins.

A wide variety of bacterial pathogens secrete a unique class of proteins that attack target cell membranes and form transmembrane oligomeric pores with distinct ß-barrel structural scaffolds. Owing to their specific mode of action and characteristic structural assembly, these proteins are termed as ß-barrel pore-forming toxins (ß-PFTs). The most obvious consequence of such pore-forming activity of bacterial ß-PFTs is the permeabilization of cell membranes, which eventually leads to cell death. Bacterial ß-PFTs have been studied extensively for nearly past four decades, and their mechanisms of actions have revealed some of the most enigmatic aspects of the protein structure-function paradigm. In most of the cases, ß-PFTs are released by the bacteria as water-soluble monomeric precursors, which upon encountering target cell membranes assemble into membrane-inserted oligomeric pores. Structural descriptions are now documented for the water-soluble precursor forms, as well as for the membrane-anchored oligomeric pores of many ß-PFTs. These studies have revealed that ß-PFTs undergo a series of well-orchestrated structural rearrangements during membrane pore formation. Nevertheless, mechanisms that trigger and regulate distinct steps of the pore-formation processes still remain obscure. Here, we discuss our current understanding regarding structure-function mechanisms in the ß-PFT family, with particular emphasis on some of the unsolved issues associated with the ß-barrel pore-formation mechanism.

Revisiting the role of cholesterol in regulating the pore-formation mechanism of Vibrio cholerae cytolysin, a membrane-damaging ß-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a ß-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.

Structural Basis and Functional Implications of the Membrane Pore-Formation Mechanisms of Bacterial Pore-Forming Toxins.

Pore-forming toxins (PFTs) are a distinct class of membrane-damaging protein toxins documented in a wide array of life forms ranging from bacteria to humans. PFTs are known to act as potent virulence factors of the bacterial pathogens. Bacterial PFTs are, in general, secreted as water-soluble molecules, which upon encountering target host cells assemble into transmembrane oligomeric pores, thus leading to membrane permeabilization and cell death. Interaction of the PFTs with the target host cells can also lead to plethora of cellular responses having critical implications for the bacterial pathogenesis processes, host-pathogen interactions, and host immunity. In this review, we present an overview of our current understanding of the structural aspects of the membrane pore-formation processes employed by the bacterial PFTs. We also discuss the functional implications of the PFT mode of actions, in terms of eliciting diverse cellular responses.