Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimer's disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self-assemble to form insoluble fibrils. X-ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross-β-scaffold, with β-strands perpendicular and β-sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol-3Ј-kinase, using cryo-electron microscopy and image processing at 25 Å resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of~600 Å and an axial subunit repeat of~27 Å. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20ϫ40-Å protofilaments can only accommodate one pair of flat β-sheets stacked against each other, with very little inter-strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.

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International audienc

The Repeat in Toxin (RTX) motif is a tandemly repeated calcium-binding nonapeptide sequence present in proteins that are secreted by the type I secretion system (T1SS) of Gram-negative bacteria. Here, we have characterized the structural and hydrodynamic properties of the RTX Repeat Domain (RD) of the CyaA toxin from Bordetella pertussis. This 701-amino acid long domain contains about 40 RTX motifs. We showed that, in the absence of calcium, RD was natively disordered, weakly stable, and highly hydrated. Calcium binding induced compaction and dehydration of RD, along with the formation of stable secondary and tertiary structures. The calcium-induced conformational switch between unfolded conformations of apo-RD and stable structures of holo-RD is likely to be a key property for the biological function of the CyaA toxin: in the low calcium environment of the bacterial cytosol, the intrinsically disordered character of the protein may facilitate its secretion through the secretion machinery. In the extracellular medium, calcium binding can then trigger the folding of the polypeptide into its functional state. The intrinsic disorder of RTX-containing proteins in the absence of calcium may thus be directly involved in the efficient secretion of proteins through T1SS.

Microcin C7 (MccC7) 1 is a small peptide antibiotic produced by Escherichia coli during the stationary phase of growth (1). The spectrum of activity of this microcin includes several members of the Enterobacteriaceae family (2). MccC7 exerts its bacteriostatic action by blocking protein synthesis. Like for other microcins, the bacterial strains that produce MccC7 are immune (resistant) to this microcin (3). The heptapeptide of MccC7 is synthesized in ribosomes (4) and undergoes posttranslational modifications to yield the mature molecule.The genetic determinants for MccC7 synthesis, export, and immunity have been cloned from the 43 kilobases E. coli pMccC7 plasmid into multicopy plasmids that overproduce MccC7 and express MccC7 immunity (5). These determinants lie on a 6.2-kilobase region of pMccC7, which has been entirely sequenced.2 Different complementary approaches, such as physical and phenotypical characterization of insertion mutations and complementation studies, have shown that this region contains six genes (mccABCDEF) (5).2 Genes A, B, C, D, and E are involved in the production of mature extracellular microcin. Genes C, E, and F code for self-immunity bestowing products. Genes mccA-D are directly involved in the synthesis and export of MccC7 and constitute an operon transcribed from a promoter (mccp), located upstream of mccA (4, 6). Expression of these genes is regulated by the cAMP-cAMP regulatory protein complex and by the stationary phase factor RpoS (also called AppR) (6, 7). 3 mccA codes for the unmodified peptide of MccC7, MRTGNAN (MccA) (4). The predicted gene polypeptide product of mccB (350 residues) is strikingly homologous to a 81-residue fragment of the ubiquitin-activating enzyme from different eucaryotic species (UBA1) (8), ThiF (9) and ChlN (10) from E. coli which participate, respectively, in the biosynthesis of thiamine pyrophosphate and of molibdopterin, and HesA, an enzyme required by Anabaena for nitrogen fixation (11). The predicted mccC product (404 residues) contains 11 potential transmembrane domains and displays significant similarity with stretches of transport proteins, 2 suggesting that MccC is responsible for MccC7 export and explaining why it also confers resistance to exogenous MccC7. The carboxyl end of the expected MccE 521-residue long polypeptide is highly similar to RimL, an enzyme that acetylates the ribosomal protein L12 from E. coli (12). Principally on the basis of this homology with an acetylating protein, and because target alteration is a common mechanism of antibiotic resistance (13), it has been proposed that MccE might confer MccC7 immunity to producing cells by acetylating the target of microcin C7.2 No similarity was found for the predicted MccD (267 residues) and MccF (334 residues) polypeptides.The knowledge of the chemical structure of MccC7 is neces-* This investigation was supported by funds from the Institut Pasteur, the Centre National de la Recherche Scientifique (URA 1129 and URA 489), and the European Union (Grant CI1*-CT92-0011 to F. M.). The...

The equilibrium unfolding and the kinetic folding and unfolding of the 67 residue Fyn-SH3 domain have been investigated. Equilibrium unfolding experiments indicate that, despite the lack of both disulfide bonds and prosthetic groups, Fyn-SH3 is relatively stable with a free energy of folding of -6.0 +/- 0.6 kcal mol-1 at 20 degrees C. Kinetic experiments indicate that the domain refolds in a rapid two-state manner without significant population of intermediates (k = 94.3 s-1 in H2O at 20 degrees C). Despite the presence of two proline residues, the refolding of the domain is monophasic, and no significant proline isomerization-like refolding phase is observed. This can be attributed to an extremely low level of the incorrect (cis) isomer of the structurally important Pro134 residue in the protein denatured in 8 M guanidine hydrochloride. Analysis of the temperature and guanidine hydrochloride dependence of the folding rate suggests that the folding transition state of this protein is relatively well organized. A comparison with the refolding kinetics and thermodynamics of other homologous SH3 domains indicates that these exhibit an equivalent degree of transition state organization. This potentially arises from conservation of key features of the transition state conformation despite sometimes relatively low overall sequence identity. Such a comparison further suggests that relative thermodynamic stability is an important factor in determining the relative folding rates of natural proteins with a common fold, but that specific details of the amino acid sequence can also play a significant role in individual cases.

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