Roman A. Melnyk

Sequence Context Strongly Modulates Association of Polar Residues in Transmembrane Helices

Polar residues are capable of mediating the association of membrane-embedded helices through the formation of side-chain/side-chain inter-helical hydrogen bonds. However, the extent to which native van der Waals packing of the residues surrounding the polar locus can enhance, or interfere with, the interaction of polar residues has not yet been studied. We examined the propensities of four polar residues (aspartic acid, asparagine, glutamic acid, and glutamine) to promote self-association of transmembrane (TM) domains in several biologically derived sequence environments, including (i). four naturally occurring TM domains that contain a Glu or Gln residue (Tnf5/CD40 ligand, C79a/Ig-alpha, C79b/Ig-beta, and Fut3/alpha-fucosyltransferase); and (ii). variants of bacteriophage M13 major coat protein TM segment with Asp and Asn at interfacial and non-interfacial positions. Self-association was quantified by the TOXCAT assay, which measures TM helix self-oligomerization in the Escherichia coli inner membrane. While an appropriately placed polar residue was found in several cases to significantly stabilize TM helix-helix interactions through the formation of an interhelical hydrogen bond, in other cases the strongly polar residues did not enhance the association of the two helices. Overall, these results suggest that an innate structural mechanism may operate to control non-specific association of membrane-embedded polar residues.

Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer's disease mutants.

BackgroundAmyloid precursor protein (APP) is enzymatically cleaved by γ-secretase to form two peptide products, either Aβ40 or the more neurotoxic Aβ42. The Aβ42/40 ratio is increased in many cases of familial Alzheimers disease (FAD). The transmembrane domain (TM) of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains.ResultsUsing synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.ConclusionThe dissociation constants are correlated to both the Aβ42/Aβ40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Aβ production and Aβ42/Aβ40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and γ-secretase components, potentially revealing a new class of γ-secretase inhibitors.

Structural Determinants for the Binding of Anthrax Lethal Factor to Oligomeric Protective Antigen

Anthrax lethal toxin assembles at the surface of mammalian cells when the lethal factor (LF) binds via its amino-terminal domain, LFN, to oligomeric forms of activated protective antigen (PA). LF·PA complexes are then trafficked to acidified endosomes, where PA forms heptameric pores in the bounding membrane and LF translocates through these pores to the cytosol. We used enhanced peptide amide hydrogen/deuterium exchange mass spectrometry and directed mutagenesis to define the surface on LFN that interacts with PA. A continuous surface encompassing one face of LFN became protected from deuterium exchange when LFN was bound to a PA dimer. Directed mutational analysis demonstrated that residues within this surface on LFN interact with Lys-197 on two PA subunits simultaneously, thereby showing that LFN spans the PA subunit:subunit interface and explaining why heptameric PA binds a maximum of three LFN molecules. Our results elucidate the structural basis for anthrax lethal toxin assembly and may be useful in developing drugs to block toxin action.

Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

Therapeutic enzyme treatment disrupts Pseudomonas biofilms, potentiating antibiotics and ameliorating the innate immune system. Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.

Crystal structure of Clostridium difficile toxin A

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Significance Clostridium difficile is the leading cause of antibiotic-associated infection in hospitals worldwide. Disease symptoms are caused by toxins A and B, which form membrane-spanning pores that deliver associated cytotoxic enzyme domains into target cells leading to cellular death and tissue damage. Despite a wealth of information for the enzymatic domains that act once inside the cell, very little is known about the translocation pore and its role in disease pathogenesis. Here we describe the structural features of the pore and identify mutants that prevent pore formation and show that they are no longer toxic to host cells. These findings offer a glimpse into the elusive translocation pore and further provide the basis for a unique strategy to target toxins therapeutically. Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues—clustered between amino acids 1,035 and 1,107—that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming “hotspots” of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.

Evidence for assembly of small multidrug resistance proteins by a "two-faced" transmembrane helix.

Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the assembly of at least two inactive monomers by intermolecular association of their four transmembrane helices. Here, we have used peptides that correspond to each of the four wild type transmembrane helices of the Halobacterium salinarum protein Hsmr and a corresponding library of mutant peptides to determine the interactive surfaces that likely contribute to protein oligomerization. Hsmr peptides were examined for strong (sodium dodecyl sulfate-resistant) and weaker (perfluorooctanoate-resistant) helix-helix interactions, in conjunction with circular dichroism, fluorescence energy transfer measurements, and molecular modeling. The results are compatible with a scheme in which two faces of helix four permit self-assembly via a higher affinity asymmetric pairing and a lower affinity symmetric interaction, resulting in a discrete tetramer. Our finding that two surfaces of helix four can contribute to the stability of small multidrug resistance protein assembly provides a molecular basis for the design of therapeutics that target this antibiotic resistance mechanism.

Cross-linked forms of the isolated N-terminal domain of the lethal factor are potent inhibitors of anthrax toxin.

ABSTRACT The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LFN, the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LFN retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20- to 50-fold more potent than monomeric LFN) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.

Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants

Clostridium difficile is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.

Clostridium difficile toxins A and B: Receptors, pores, and translocation into cells

Abstract The most potent toxins secreted by pathogenic bacteria contain enzymatic moieties that must reach the cytosol of target cells to exert their full toxicity. Toxins such as anthrax, diphtheria, and botulinum toxin all use three well-defined functional domains to intoxicate cells: a receptor-binding moiety that triggers endocytosis into acidified vesicles by binding to a specific host-cell receptor, a translocation domain that forms pores across the endosomal membrane in response to acidic pH, and an enzyme that translocates through these pores to catalytically inactivate an essential host cytosolic substrate. The homologous toxins A (TcdA) and Toxin B (TcdB) secreted by Clostridium difficile are large enzyme-containing toxins that for many years have eluded characterization. The cell-surface receptors for these toxins, the non-classical nature of the pores that they form in membranes, and mechanism of translocation have remained undefined, exacerbated, in part, by the lack of any structural information for the central ∼1000 amino acid translocation domain. Recent advances in the identification of receptors for TcdB, high-resolution structural information for the translocation domain, and a model for the pore have begun to shed light on the mode-of-action of these toxins. Here, we will review TcdA/TcdB uptake and entry into mammalian cells, with focus on receptor binding, endocytosis, pore formation, and translocation. We will highlight how these toxins diverge from classical models of translocating toxins, and offer our perspective on key unanswered questions for TcdA/TcdB binding and entry into mammalian cells.