Atomic View of a Toxic Amyloid Small Oligomer

See allHide authors and affiliations

Science  09 Mar 2012:
Vol. 335, Issue 6073, pp. 1228-1231
DOI: 10.1126/science.1213151

You are currently viewing the figures only.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

  1. Fig. 1

    The cylindrins derived from ABC, an amyloid-forming protein, exhibit the properties of oligomeric state, immunoreactivity, and cytotoxicity commonly ascribed to small amyloid oligomers. (A) Ribbon diagram of a single subunit of ABC (16), colored by propensity to form amyloid, with red being the highest and blue the lowest propensity. The segment from residue 90 to 100, termed K11V, forms the cylindrin. (B) Representative electron micrograph of amyloid fibrils formed by K11V-TR. (C) Overlaid size-exclusion chromatograms showing protein standards (blue dashed curve) and cylindrin segments. K11VV2L (purple curve; 1.2 kD per chain) and K11V-TR (green curve; 2.5 kD per chain) cylindrin segments migrate as oligomeric complexes. A mutant form of K11V-TR that disrupts oligomer formation of the cylindrin peptide, K11VV4W-TR (orange curve; 2.7 kD per chain), migrates as a dimeric or monomeric species. (D) Native nanoelectrospray mass spectrum of K11V-TR peak fractions from SEC-HPLC reveals trimeric tandem-repeat cylindrin oligomers, confirming that the oligomeric complexes coincide in mass with the crystallized cylindrins. Expansion of the most abundant ion series of a +5 charge state corresponding to a molecular mass of three K11V-TR chains, coinciding with the crystallographic trimeric oligomer with a mass accuracy of 3.93 parts per million (ppm), is shown, with mass/charge (m/z) labels. (E) Immunodot blot analysis of solutions of K11VV2L and K11V-TR oligomers and K11V-TR fibrils with prefibrillar oligomer-specific, polyclonal antibody A11 (5); and a mixture of fibril-specific monoclonal antibodies, OC (11). Solutions of cylindrin-forming segments are recognized by A11 but not by the OC antibody. In contrast, K11V-TR fibrils are recognized only by the OC antibody. Positive controls are shown to the right (5). (F) Cylindrin K11V-TR is toxic to four mammalian cell lines. Cell viability levels return to nearly 100% when we tested the control variant K11VV4W-TR. All samples were at a final concentration of 100 μM. Results represent mean ± SEM. Student’s t test (N = 4): **P < 0.01; ***P < 0.001.

  2. Fig. 2

    Crystal structures of cylindrins and computed free energy change of the simulated structural transition from cylindrin to a fibril. Each colored β strand (arrow) is composed of 11 amino acid residues from ABC of sequence KVKVLGDVIEV (K11V). (A) Schematic of unrolled cylindrin (outside view), illustrating strand-to-strand registration. Hydrogen bonds between the main chains of neighboring strands are shown by yellow dashed lines; hydrogen bonds mediated by water bridges or side chains are shown by blue dashed lines. (B) Ribbon representation of the cylindrin crystal structure. Pairs of strands form antiparallel dimers, which assemble around a threefold axis down the barrel axis of the cylindrin. The height of the cylindrin is 22 Å. The inner dimension of the cylindrin, around the waist from Cα to Cα, is 12 Å, and at the splayed ends the diameter is 22 Å. (C) The cylindrin with side chains shown as atoms and hydrogen bonds in yellow. Twelve backbone hydrogen bonds stabilize the strong interface between tightly twisted antiparallel strands (e.g., between green and purple chains). The weaker interface between the pairs of tightly twisted strands is formed by four main-chain hydrogen bonds, with an additional two hydrogen bonds coming from a water bridge and two hydrogen bonds from side-chain interactions (e.g., between purple and blue chains). The dry interior of the cylinder is closed by triplets of Val residues, shown as spheres, at the top and bottom. (D) Crystal structure of K11V-TR formed by three chains of 25 residues each. (E) Schematic of unrolled K11V-TR cylindrin (outside view). Similar hydrogen-bonding patterns are formed as in (A). (F) The computed Gibbs free energy at 300 K for a cylindrin forced to a fibril. The reaction coordinate measures the difference in root mean square deviation (ΔRMSD) from the two end points: the cylindrin and the in-register antiparallel β sheet (IAB). The cylindrin set the free energy minimum (point 1). The transition was initiated by disrupting the weak interface (points 2 and 3). As the cylindrin unrolls, the weak interface requires complete dissociation of backbone hydrogen bonds (points 4 and 5), whereas the strong interfaces maintains hydrogen bonding (point 6). The IAB has a higher free energy than the cylindrin (point 7), and when two IABs associate and interdigitate to form a steric zipper (point 8) the free energy drops to 5.2 kcal/mol per peptide lower than the cylindrin (table S4).

Stay Connected to Science