Structural studies of T4S systems by electron microscopy

Abstract: Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNA and/or proteins through the membranes of bacteria. Analysis of T4S system architecture is an extremely challenging task taking into account their multi protein organisation and lack of overall global symmetry. Nonetheless the last decade demonstrated an amazing progress achieved by X-ray crystallography and cryo-electron microscopy. In this review we present a structural analysis of this dynamic complex based on recent advances in biochemical, biophysical and structural studies

Structural basis of pore formation by the bacterial toxin pneumolysin.

The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM and image processing, we have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts beta hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 A in diameter lined by 176 beta strands

Dodecameric structure of the small heat shock protein Acr1 from Mycobacterium tuberculosis.

Small heat shock proteins are a ubiquitous and diverse family of stress proteins that have in common an alpha-crystallin domain. Mycobacterium tuberculosis has two small heat shock proteins, Acr1 (alpha-crystallin-related protein 1, or Hsp16.3/16-kDa antigen) and Acr2 (HrpA), both of which are highly expressed under different stress conditions. Small heat shock proteins form large oligomeric assemblies and are commonly polydisperse. Nanoelectrospray mass spectrometry showed that Acr2 formed a range of oligomers composed of dimers and tetramers, whereas Acr1 was a dodecamer. Electron microscopy of Acr2 showed a variety of particle sizes. Using three-dimensional analysis of negative stain electron microscope images, we have shown that Acr1 forms a tetrahedral assembly with 12 polypeptide chains. The atomic structure of a related alpha-crystallin domain dimer was docked into the density to build a molecular structure of the dodecameric Acr1 complex. Along with the differential regulation of these two proteins, the differences in their quaternary structures demonstrated here supports their distinct functional roles

Elongated oligomers assemble into mammalian PrP Amyloid fibrils

In prion diseases, the mammalian prion protein PrP is converted from a monomeric, mainly α-helical state into β-rich amyloid fibrils. To examine the structure of the misfolded state, amyloid fibrils were grown from a β form of recombinant mouse PrP (residues 91–231). The β-PrP precursors assembled slowly into amyloid fibrils with an overall helical twist. The fibrils exhibit immunological reactivity similar to that of ex vivo PrPSc. Using electron microscopy and image processing, we obtained three-dimensional density maps of two forms of PrP fibrils with slightly different twists. They reveal two intertwined protofilaments with a subunit repeat of not, vert, similar60 Å. The repeating unit along each protofilament can be accounted for by elongated oligomers of PrP, suggesting a hierarchical assembly mechanism for the fibrils. The structure reveals flexible crossbridges between the two protofilaments, and subunit contacts along the protofilaments that are likely to reflect specific features of the PrP sequence, in addition to the generic, cross-β amyloid fold

Atomic structure and hierarchical assembly of a cross-β amyloid fibril

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale--including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy--we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils