Suren A. Tatulian

Effect of lipid phase transition on the binding of anions to dimyristoylphosphatidylcholine liposomes

Temperature dependence of the electrophoretic mobility of multilamellar liposomes prepared from dimyristoylphosphatidylcholine was measured in the presence of salts with different anions in aqueous solutions. It was established that specific binding of anions to liposome surface induced a pronounced zeta potential (electrostatic potential at the hydrodynamic plane of shear). A combination of Langmuir, Gouy-Chapman, and Boltzmann equations was used to describe the dependence of the zeta potential on the concentration of anions. The values of binding constants (K) and maximum numbers of binding sites per unit area (sigma max) were determined by this method. The sequence for anion affinities to liposome surface was found to be as follows: trinitrophenol greater than ClO-4 greater than I- greater than SCN- greater than Br- greater than NO-3 greater than Cl- approximately or equal to SO2-4. A sharp increase in the negative zeta potential was detected at the temperature of phase transition of the lipid from the gel to liquid-crystalline state. It was found that the parameter K did not change at lipid phase transition and the shifts in zeta potential might be due to alterations of sigma max. The binding sites were considered as defects in the package of lipid molecules in membranes.

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

The phospholipase A2 (PLA2) enzymes are activated by binding to phospholipid membranes. Although the N-terminal α-helix of group I/II PLA2s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA2s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA2, we have created semisynthetic human group IB PLA2 in which the N-terminal decapeptide is joined with the 13C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA2 joined with a 13C-labeled fragment of group IB PLA2. Infrared spectral resolution of the unlabeled and 13C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA2 has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA2 is more rigid than that of the IB PLA2, but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA2, which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA2. Correlation is delineated between structural and membrane binding properties of PLA2s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA2s acts as a regulatory domain that mediates interfacial activation of these enzymes.

Stabilization of the Tertiary Structure of the Cholera Toxin A1 Subunit Inhibits Toxin Dislocation and Cellular Intoxication

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic subunit of CT (CTA1) then crosses the ER membrane and enters the cytosol in a process that involves the quality control mechanism of ER-associated degradation. The molecular details of this dislocation event have not been fully characterized. Here, we report that thermal instability in the CTA1 subunit-specifically, the loss of CTA1 tertiary structure at 37 degrees C-triggers toxin dislocation. Biophysical studies found that glycerol preferentially stabilized the tertiary structure of CTA1 without having any noticeable effect on the thermal stability of its secondary structure. The thermal disordering of CTA1 tertiary structure normally preceded the perturbation of its secondary structure, but in the presence of 10% glycerol the temperature-induced loss of CTA1 tertiary structure occurred at higher temperatures in tandem with the loss of CTA1 secondary structure. The glycerol-induced stabilization of CTA1 tertiary structure blocked CTA1 dislocation from the ER and instead promoted CTA1 secretion into the extracellular medium. This, in turn, inhibited CT intoxication. Glycerol treatment also inhibited the in vitro degradation of CTA1 by the core 20S proteasome. Collectively, these findings indicate that toxin thermal instability plays a key role in the intoxication process. They also suggest the stabilization of CTA1 tertiary structure is a potential goal for novel antitoxin therapeutic agents.

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Protein-disulfide isomerase (PDI) has been proposed to exhibit an “unfoldase” activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin.

A novel mode of translocation for cytolethal distending toxin.

Thermal instability in the toxin catalytic subunit may be a common property of toxins that exit the endoplasmic reticulum (ER) by exploiting the mechanism of ER-associated degradation (ERAD). The Haemophilus ducreyi cytolethal distending toxin (HdCDT) does not utilize ERAD to exit the ER, so we predicted the structural properties of its catalytic subunit (HdCdtB) would differ from other ER-translocating toxins. Here, we document the heat-stable properties of HdCdtB which distinguish it from other ER-translocating toxins. Cell-based assays further suggested that HdCdtB does not unfold before exiting the ER and that it may move directly from the ER lumen to the nucleoplasm. These observations suggest a novel mode of ER exit for HdCdtB.

A Therapeutic Chemical Chaperone Inhibits Cholera Intoxication and Unfolding/Translocation of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels as an intact AB5 protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera.

Contribution of Subdomain Structure to the Thermal Stability of the Cholera Toxin A1 Subunit

The catalytic A1 subunit of cholera toxin (CTA1) is an ADP-ribosyltransferase with three distinct subdomains: CTA1(1) forms the catalytic core of the toxin, CTA1(2) is an extended linker between CTA1(1) and CTA1(3), and CTA1(3) is a compact globular region. CTA1 crosses the endoplasmic reticulum (ER) membrane to enter the cytosol where it initiates a cytopathic effect. Toxin translocation involves ER-associated degradation (ERAD), a quality control system that exports misfolded proteins from the ER to the cytosol. At the physiological temperature of 37 °C, the free CTA1 subunit is in a partially unfolded conformation that triggers its ERAD-mediated translocation to the cytosol. Thus, the temperature sensitivity of CTA1 structure is an important determinant of its function. Here, we examined the contribution of CTA1 subdomain structure to the thermal unfolding of CTA1. Biophysical measurements demonstrated that the CTA1(1) subdomain is thermally unstable and that the CTA1(2) subdomain provides a degree of conformational stability to CTA1(1). The CTA1(3) subdomain does not affect the overall stability of CTA1, but the thermal unfolding of CTA1 appears to begin with a local loss of structure in the CTA1(3) subdomain: glycerol and acidic pH both inhibited the thermal disordering of full-length CTA1 but not the disordering of a CTA1 construct lacking the A1(3) subdomain. These observations provide mechanistic insight regarding the thermal unfolding of CTA1, an event which facilitates its subsequent translocation to the cytosol.

The pertussis toxin S1 subunit is a thermally unstable protein susceptible to degradation by the 20S proteasome.

Pertussis toxin (PT) is an AB-type protein toxin that consists of a catalytic A subunit (PT S1) and an oligomeric, cell-binding B subunit. It belongs to a subset of AB toxins that move from the cell surface to the endoplasmic reticulum (ER) before the A chain passes into the cytosol. Toxin translocation is thought to involve A chain unfolding in the ER and the quality control mechanism of ER-associated degradation (ERAD). The absence of lysine residues in PT S1 may allow the translocated toxin to avoid ubiquitin-dependent degradation by the 26S proteasome, which is the usual fate of exported ERAD substrates. As the conformation of PT S1 appears to play an important role in toxin translocation, we used biophysical and biochemical methods to examine the structural properties of PT S1. Our in vitro studies found that the isolated PT S1 subunit is a thermally unstable protein that can be degraded in a ubiquitin-independent fashion by the core 20S proteasome. The thermal denaturation of PT S1 was inhibited by its interaction with NAD, a donor molecule used by PT S1 for the ADP ribosylation of target G proteins. These observations support a model of intoxication in which toxin translocation, degradation, and activity are all influenced by the heat-labile nature of the isolated toxin A chain.

Structural Effects of Covalent Inhibition of Phospholipase A2 Suggest Allosteric Coupling between Membrane Binding and Catalytic Sites

Phospholipase A(2) (PLA(2)) binds to membranes and catalyzes phospholipid hydrolysis, thus initiating the biosynthesis of lipid-derived mediators of inflammation. A snake-venom PLA(2) was completely inhibited by covalent modification of the catalytic histidine 48 by p-bromophenacyl bromide. Moreover, His(48) modification affected PLA(2) structure, its membrane-binding affinity, and the effects of PLA(2) on the membrane structure. The native PLA(2) increased the order parameter of fluid membranes, whereas the opposite effect was observed for gel-state membranes. The data suggest membrane dehydration by PLA(2) and the formation of PLA(2)-membrane hydrogen bonding. The inhibited PLA(2) had lower membrane-binding affinity and exerted weaker effects on membrane hydration and on the lipid-order parameter. Although membrane binding resulted in formation of more flexible alpha-helices in the native PLA(2), which corresponds to faster amide hydrogen exchange, the modified enzyme was more resistant to hydrogen exchange and experienced little structural change upon membrane binding. The data suggest that 1), modification of a catalytic residue of PLA(2) induces conformational changes that propagate to the membrane-binding surface through an allosteric mechanism; 2), the native PLA(2) acquires more dynamic properties during interfacial activation via membrane binding; and 3), the global conformation of the inhibited PLA(2), including the alpha-helices, is less stable and is not influenced by membrane binding. These findings provide further evidence for an allosteric coupling between the membrane-binding (regulatory) site and the catalytic center of PLA(2), which contributes to the interfacial activation of the enzyme.

Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit

Background: Cholera toxin enters the target cell in a disordered state and must attain a folded, active conformation to modify its G protein target. Results: Lipid rafts, where G protein is located, shift the disordered toxin to a functional conformation. Conclusion: Lipids rafts provide a chaperone-like function for cholera toxin. Significance: Lipid rafts play an important role in regulating toxin function through chaperone-like activity. Cholera toxin (CT) travels from the cell surface to the endoplasmic reticulum (ER) as an AB holotoxin. ER-specific conditions then promote the dissociation of the catalytic CTA1 subunit from the rest of the toxin. CTA1 is held in a stable conformation by its assembly in the CT holotoxin, but the dissociated CTA1 subunit is an unstable protein that spontaneously assumes a disordered state at physiological temperature. This unfolding event triggers the ER-to-cytosol translocation of CTA1 through the quality control mechanism of ER-associated degradation. The translocated pool of CTA1 must regain a folded, active structure to modify its G protein target which is located in lipid rafts at the cytoplasmic face of the plasma membrane. Here, we report that lipid rafts place disordered CTA1 in a functional conformation. The hydrophobic C-terminal domain of CTA1 is essential for binding to the plasma membrane and lipid rafts. These interactions inhibit the temperature-induced unfolding of CTA1. Moreover, lipid rafts could promote a gain of structure in the disordered, 37 °C conformation of CTA1. This gain of structure corresponded to a gain of function: whereas CTA1 by itself exhibited minimal in vitro activity at 37 °C, exposure to lipid rafts resulted in substantial toxin activity at 37 °C. In vivo, the disruption of lipid rafts with filipin substantially reduced the activity of cytosolic CTA1. Lipid rafts thus exhibit a chaperone-like function that returns disordered CTA1 to an active state and is required for the optimal in vivo activity of CTA1.