Rapid Topology Determination of Membrane Proteins: Pore-Forming Mechanism of Bt toxin Cry1Aa

Abstract

While crystal structures provide a structure with atomic resolution, it is only limitedly applicable to membrane toxins. The reason is that the mechanism of the toxins is based on a complete refolding of the protein ones it incorporates into the membrane. Cry1Aa, a toxin of Bacillus thuringiensis, is a widely used biological pesticide. It has been shown that the toxin consists of three structural distinct domains. While domains II and III are involved in receptor binding and host specificity, domain I forms cation-selective pores in bilayer membranes. The present study aims to investigate the topology of the toxin at the membrane previous to the pore formation and the structural determinants of the pore forming mechanism of Bt toxin. Previous models predicted that the toxin anneals to the surface of the membrane before triggering the pore formation by inserting the α-helices 4-5 through the membrane, while the other helices are thought to remain on the external leaflet (Vachon et al., 2003). We labelled the toxin by site-directed fluorescence labelling and inserted it into horizontal planar lipid bilayer. We performed FRET measurements between these distinct positions in the toxin and the bilayer. This way, we could determine the location of the residues with respect to the centerline of the membrane and create a topology map of the protein. In contrast to the previous model, we found that most of domain I translocates through the membrane upon insertion and accumulates in the inner leaflet. Preliminary data suggest that for pore formation the helices 3 and 4 translocate through the membrane from the inner to the outer leaflet. This is consitent with helix 4 being the pore-lining helix as suggested by electrophysiological data.Supported by NSERC-327201DG