Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host-pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.
The HIV surface glycoprotein, Env, initiates HIV infection by binding to cellsurface CD4 and a co-receptor molecule on T cells. Understanding the interactions of these molecules with native, trimeric Env is crucial for effective vaccine design. using cryo-electron tomography of intact HIV, we show that binding to either the co-receptor mimic 17b or soluble CD4 is sufficient for formation of an open quaternary conformation of Env. In contrast, binding of the broadly neutralizing, CD4-binding-site antibody, VRC01, locks Env in the native, closed conformation, preventing 17b binding and formation of the open Env conformation. The CD4-binding-site antibody, b12, requires a partial opening in the quaternary structure of Env and cannot bind in the conformation that binds to the VRC antibodies due to steric clashes. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01 and b12, important differences exist in the quaternary structures of these complexes. These findings shed new mechanistic insight into the HIV entry process and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities.
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