Orientation in Lipid Bilayers of a Synthetic Peptide Representing the C-Terminus of the A₁ Domain of Shiga Toxin. A Polarized ATR-FTIR Study

Abstract

The interaction of a synthetic peptide representing the C-terminal 27 amino acids of the A1 domain of Shiga toxin (residues 220-246) with acidic phospholipid model membranes was characterized by FTIR spectroscopy. This peptide resembles a signal sequence and may mediate the translocation of the catalytic A1 chain of Shiga toxin to the cytoplasm following its retrograde transport to the lumenal compartment of the endoplasmic reticulum (ER). At pH 7 and 5, the peptide underwent a conformational change from random coil to alpha-helix upon addition of negatively charged phospholipids. Examination of the amide II band in the spectrum of the complex at pH 7 and pH 5 showed that in both cases, the N-H groups in the peptide backbone are largely protected from H/D exchange. Using polarized attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) measurements, the orientation of the alpha-helical portion of the peptide was found to be almost perpendicular with respect to the membrane plane at pH 7. However, at pH 5.0-5.4, the alpha-helix axis was preferentially oriented parallel to the membrane plane. The results suggest that at the neutral pH of the ER lumen, the peptide may insert into the membrane, while at the lower pH levels present in earlier endocytic compartments, the peptide would be less likely to traverse the bilayer. In summary, this putative signal peptide may not be able to cause a significant translocation of the A1 domain of Shiga toxin to the cytosol until it reaches the neutral pH of the ER compartment.

Transmembrane orientation and possible role of the fusogenic peptide from parainfluenza virus 5 (PIV5) in promoting fusion

“…The dichroic ratio for parallel versus perpendicularly polarized light was 3.2 and 3.6 for the wild type and mutant, respectively. These values correspond to an orientation of approximately 29°and 22°relative to the membrane normal (67,68), assuming the entire peptide is fully helical and the bilayers are well ordered. Deviation from helical geometry or disorder of the bilayer would result in somewhat lower dichroic ratios.…”

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

“…Thus, although it is not required for ERAD-mediated toxin translocation (54), the A1 3 subdomain still plays a functional role in host-toxin interactions. C-terminal domains from the catalytic subunits of Shiga toxin and ricin, two other ER-translocating toxins, also interact with lipid bilayers and play important roles in intoxication (62)(63)(64)(65). For ricin A chain, the C terminus has been shown to mediate an interaction with the negatively charged phospholipids of the ER membrane which results in toxin unfolding (62,63).…”

How Ricin and Shiga Toxin Reach the Cytosol of Target Cells: Retrotranslocation from the Endoplasmic Reticulum

“…Furthermore, an artificial peptide based on this sequence interacts with lipid membranes (Menikh et al 1997;Saleh et al 1996). Whilst the parellels with RTA structure might seem obvious, we note that this region is already substantially exposed in the holotoxin (Figure 4, blue), and it remains to be determined whether membrane interactions induce conformational changes in SLTxA1 that are then recognized by the dislocation machinery.…”