Protective antigen (PA) is the central component of the three-part protein toxin secreted by Bacillus anthracis, the organism responsible for anthrax. After proteolytic activation on the host cell surface, PA forms a membrane-inserting heptamer that translocates the toxic enzymes, oedema factor and lethal factor, into the cytosol. PA, which has a relative molecular mass of 83,000 (M(r) 83K), can also translocate heterologous proteins, and is being evaluated for use as a general protein delivery system. Here we report the crystal structure of monomeric PA at 2.1 A resolution and the water-soluble heptamer at 4.5 A resolution. The monomer is organized mainly into antiparallel beta-sheets and has four domains: an amino-terminal domain (domain 1) containing two calcium ions and the cleavage site for activating proteases; a heptamerization domain (domain 2) containing a large flexible loop implicated in membrane insertion; a small domain of unknown function (domain 3); and a carboxy-terminal receptor-binding domain (domain 4). Removal of a 20K amino-terminal fragment from domain 1 allows the assembly of the heptamer, a ring-shaped structure with a negatively charged lumen, and exposes a large hydrophobic surface for binding the toxic enzymes. We propose a model of pH-dependent membrane insertion involving the formation of a porin-like, membrane-spanning beta-barrel.

Cytosolic proteins bearing a classical nuclear localization signal enter the nucleus bound to a heterodimer of importin-alpha and importin-beta (also called karyopherin-alpha and -beta). The formation of this heterodimer involves the importin-beta-binding (IBB) domain of importin-alpha, a highly basic amino-terminal region of roughly 40 amino-acid residues. Here we report the crystal structure of human importin-beta bound to the IBB domain of importin-alpha, determined at 2.5 A and 2.3 A resolution in two crystal forms. Importin-beta consists of 19 tandemly repeated HEAT motifs and wraps intimately around the IBB domain. The association involves two separate regions of importin-beta, recognizing structurally distinct parts of the IBB domain: an amino-terminal extended moiety and a carboxy-terminal helix. The structure indicates that significant conformational changes occur when importin-beta binds or releases the IBB domain domain and suggests how dissociation of the importin-alpha/beta heterodimer may be achieved upon nuclear entry.

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The 14-3-3 family of proteins have recently been identified as regulatory elements in intracellular signalling pathways: 14-3-3 proteins bind to oncogene and proto-oncogene products, including c-Raf-1 (refs 2-5), c-Bcr (ref. 6) and polyomavirus middle-T antigen; overexpression of 14-3-3 activates Raf kinase in yeast and induces meiotic maturation in Xenopus oocytes. Here we report the crystal structure of the major isoform of mammalian 14-3-3 proteins at 2.9 A resolution. Each subunit of the dimeric protein consists of a bundle of nine antiparallel helices that form a palisade around an amphipathic groove. The groove is large enough to accommodate a tenth helix, and we propose that binding to an amphipathic helix represents a general mechanism for the interaction of 14-3-3 with diverse cellular proteins. The residues in the dimer interface and the putative ligand-binding surface are invariant among vertebrates, yeast and plants, suggesting a conservation of structure and function throughout the 14-3-3 family.