Structure and amyloid formation of hβ2M and mβ2M. (A) In the ribbon diagram of the hβ2M structure (PDB 1LDS), the segments of identical sequence in hβ2M and mβ2M are shown in dark gray. The amyloid-forming hβM7mer (within the shaded circle) is the loop nearest to the C terminus, connecting β-strands F and G. The disulfide bond between Cys 25 and Cys 80 is shown as ball-and-stick. (B) The alignment of the sequences shows that hβ2M and mβ2M are 70% identical. Residues 83–89 (in the oval) form the longest segment, in which the hβ2M and mβ2M sequences differ ( indicates β-strands; indicates the β-hairpin). (C) The amyloid-forming properties of hβ2M and mβ2M. hβ2M forms amyloid (filled circles), as judged by the characteristic fluorescence after staining the sample with ThT. In contrast, under the same conditions, mβM forms amorphous aggregates (open circles) and does not bind ThT [drawn with molscript (35) and secseq (http://xray.imsb.au.dk/~deb/secseq)].

Amyloid fiber formation by hβ2M7mer. (A) hβ2M7mer fibrils stained with Congo red solution appear red when viewed under unpolarized light. (B) When viewed between crosspolarizers, the sample shown in A exhibits “apple green” birefringence typical for amyloid fibrils. (C) Samples of hβM7mer contain fibrils when viewed by EM.

Speculative Zipper-spine model for the β2M protofilament. (A) The C-terminal segment of β2M is the portion of the structure to rearrange during fibril formation and the amyloid-forming hβ2M7mer (boxed) forms a new β-strand F′.(B) In the fibril model, residues Pro 90 and Lys 91 form a type I β-turn. Thus the β-strand G is hydrogen bonded with the new β-strand F′ rather than with the β-strand F of the native structure. (C) Two such F′-G hairpins stack to form the asymmetric unit of one of the sheets of the spine. Each sheet is built with a small (7°) twist between β-strands, stacked 4.7 Å apart, so that the spine has a pitch of ≈242 Å. We expect these parameters will be refined as structural data become available. The sheets are separated by ≈11 Å. β-Strands A–E and part of F retain their native structure with the disulfide bond between β-strands B and F intact. These native-like core domains decorate the periphery of the double β-sheet spine, with only four molecules shown for clarity. (D) The view down the fiber axis shows the double β-sheet spine. The rest of the β2M molecules, which remain folded, are packed closely around the β-sheet spine. (E) A space-filling model (view of the fibril down its axis) shows that each of the four molecules, represented with different colors, is tightly packed within the fibril, with no space for solvent. Thus, the core domains shield the spine from solvent.

Amyloid fibril formation of wild-type and chimeric β2M. (A) mβ2M/wt (filled circles) does not bind ThT. In contrast, the mβ2M/h aggregates (open circles) bind ThT starting ≈4 days after initiating the reaction. This demonstrates the ability of the human heptamer to promote mβ2M to form amyloid-like aggregates. (B) The mβ2M/h sample consists of long straight fibrils typical for amyloids. (C) No fibrils were observed in the mβ2M/wt sample, and the largest aggregates observed appear globular, as indicated by the white arrows. Micrographs shown in B and C were taken 14 days after the initiation of the aggregation reaction. (D) Fibril formation of hβ2M is disrupted when the mβ2M7mer is swapped into the hβ2M scaffold. Aggregation of the hβ2M/m chimera (open circles) lags behind that of the hβ2M/wt (filled circles) fibril formation. hβ2M/wt reaches the aggregated state ≈3–24 h earlier than hβ2M/m. The fluorescence of the two proteins after the aggregated state is reached is indistinguishable. (E) hβ2M/wt sample had a homogenous population of long and straight fibrils, typical for amyloid, and no amorphous aggregates were observed in the sample. (F) In contrast, hβ2M/m sample displayed equal populations of long (white arrow) and short fibrils (black arrow). The electron micrographs of samples shown in E and F were taken 10 days after the initiation of the aggregation reaction.

X-ray diffraction cross β pattern of β2M/wt fibrils. (A) The characteristic reflections for amyloid fibrils are indicated by arrows. (B) Comparison of the observed profile of the ≈11-Å diffuse reflection with profiles calculated from models representing the Zipper-spine as one to four slits (β-sheets).