Dept. of Microbiology and
Harvard Medical School
NRB, Room 1037 A
77 Avenue Louis Pasteur
Boston, MA 02115
Toxic proteins produced by pathogenic bacteria contribute in major ways to the symptoms of diseases caused by the bacteria. The most potent bacterial toxins enter mammalian cells and covalently modify target substrates within the cytosol. Well known examples are diphtheria, anthrax, cholera, tetanus, and botulinum toxins. The primary benefit to the bacteria of producing such toxins is thought to be, in most cases, disruption of key functions of cells of the host’s immune system, thereby protecting the bacteria from immune destruction.
The Collier group has traditionally focused on bacterial toxins that modify cytosolic targets, the goal being a detailed understanding of toxin structure and mode of action of these proteins at the biochemical and cellular levels. For all intracellularly acting toxins a catalytically active subunit must cross a membrane to gain access to the cytosol. In most such toxins the catalytic function is carried by a separate polypeptide (the “A” chain) from the receptor binding function (the “B” chain), and in some toxins the B chain also has the capacity to form a transmembrane pore (channel) that mediates transmembrane translocation of the A chain. We have been investigating the structures of the pores formed and how they function in translocation. In the past we studied translocation diphtheria toxin, in which an α-helical domain (T domain) within the B chain forms a pore that is somehow related to this process. In recent years most of our effort has been focused on anthrax toxin, a more tractable system, which has yielded a more detailed understanding of pore structure and function.
Anthrax toxin is comprised of three monomeric proteins: Lethal Factor (LF; a 90 kDa metalloprotease), Edema Factor (EF; an 89 kDa adenylate cyclase), and Protective Antigen (PA; an 83 kDa receptor-binding, pore-forming protein). After being proteolytically activated, PA self-associates to form heptameric and octameric ring-shaped oligomers, which in turn bind LF and EF. These receptor-bound complexes are endocytosed transported to the endosomal compartment within cells, where acidic conditions convert the PA oligomers into pores that translocate LF and EF across the membrane to the cytosol.
Currently we are conducting studies to understand in great detail how the PA pores function in translocation. Our data indicate that the pore is an active transporter, in which the pH gradient across the endosomal membrane may be the primary driving force for translocation. We believe translocation occurs by a Brownian ratchet mechanism, in which ratcheting (directionally biased diffusion) of substrate proteins (LF and EF) through the pore occurs in response to preferential protonation of acidic side chains of the substrate in the endosomal compartment.
Krantz BA, Melnyk RA, Zhang S, Juris SJ, Lacy DB, Wu A, Finkelstein A, and RJ Collier. 2005. A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore. Science 309:777-781.
Krantz BA, Finkelstein A and RJ Collier. 2006. Protein translocation through anthrax toxin’s transmembrane pore is driven by a proton gradient. J Mol Biol, 355:968-979.
Young JAT and RJ Collier. 2007. Anthrax toxin: Receptor binding, internalization, pore formation, and translocation. Ann Rev Biochem 76:243-65.
Sun J, Lang A, Aktories K and RJ Collier. 2008. Phenylalanine-427 of anthrax protective antigen functions in both pore formation and translocation. Proc Natl Acad Sci U S A, 105:4346-4351.
Vernier G, Wang J, Jennings LD, Sun J, Fischer A, Song L and RJ Collier. 2009. Solubilization and characterization of the anthrax toxin pore in detergent micelles. Protein Science 18:1882-95.
Janowiak BE, Fischer A, and RJ Collier. 2010. Effects of introducing a single charged residue into the Phe clamp of multimeric protective antigen. J Biol Chem [Epub ahead of print]
Katayama H, Wang J, Tama F, Chollet L, Gogol EP, Collier RJ, and MT Fisher. 2010. Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles. Proc Natl Acad Sci USA 107:3453-7.
Pentelute BL, Barker AP, Janowiak BE, Kent SBH, and RJ Collier. 2010. A semisynthesis platform for investigating structure-function relationships in the N-terminal domain of the anthrax lethal factor. ACS Chem Biol [Epub ahead of print]