A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins.

The cholesterol-dependent cytolysins (CDCs) are bacterial, ß-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs.IMPORTANCE The cholesterol-dependent cytolysins' pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the ß-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease.

The Structural Basis for a Transition State That Regulates Pore Formation in a Bacterial Toxin.

The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning ß-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (Ea) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other ß-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation.IMPORTANCE The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning ß-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large ß-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species.

Structural Basis for Receptor Recognition by the Human CD59-Responsive Cholesterol-Dependent Cytolysins.

Cholesterol-dependent cytolysins (CDCs) are a family of pore-forming toxins that punch holes in the outer membrane of eukaryotic cells. Cholesterol serves as the receptor, but a subclass of CDCs first binds to human CD59. Here we describe the crystal structures of vaginolysin and intermedilysin complexed to CD59. These studies, together with small-angle X-ray scattering, reveal that CD59 binds to each at different, though overlapping, sites, consistent with molecular dynamics simulations and binding studies. The CDC consensus undecapeptide motif, which for the CD59-responsive CDCs has a proline instead of a tryptophan in the motif, adopts a strikingly different conformation between the structures; our data suggest that the proline acts as a selectivity switch to ensure CD59-dependent CDCs bind their protein receptor first in preference to cholesterol. The structural data suggest a detailed model of how these water-soluble toxins assemble as prepores on the cell surface.

Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation.

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world's leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.

Manipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin.

The cholesterol-dependent cytolysins (CDCs) attack cells by punching large holes in their membranes. Lectinolysin from Streptococcus mitis is unique among CDCs due to the presence of an N-terminal lectin domain that enhances the pore-forming activity of the toxin. We recently determined the crystal structures of the lectin domain in complex with various glycans. These structures revealed the molecular basis for the Lewis antigen specificity of the toxin. Based on this information we have used in silico molecular modeling to design a mutant toxin, which we predicted would increase its specificity for Lewis y, an antigen found on the surface of cancer cells. Surprisingly, we found by surface plasmon resonance binding experiments that the resultant mutant lectin domain exhibited higher specificity for Lewis b antigens instead. We then undertook comparative crystallographic and molecular dynamics simulation studies of the wild-type and mutant lectin domains to understand the molecular basis for the disparity between the theoretical and experimental results. The crystallographic results revealed that the net number of interactions between Lewis y and wild-type versus mutant was unchanged whereas there was a loss of a hydrogen bond between mutant and Lewis b compared to wild-type. In contrast, the molecular dynamics studies revealed that the Lewis b antigen spent more time in the binding pocket of the mutant compared to wild-type and the reverse was true for Lewis y. The results of these simulation studies are consistent with the conclusions drawn from the surface plasmon resonance studies. This work is part of a program to engineer lectinolysin so that it will target and kill specific cells in human diseases.