3D structure of amyloid protofilaments of β ₂ -microglobulin fragment probed by solid-state NMR

Abstract

Understanding the structure and formation of amyloid fibrils, the filamentous aggregates of proteins and peptides, is crucial in preventing diseases caused by their deposition and, moreover, for obtaining further insight into the mechanism of protein folding and misfolding. We have combined solid-state NMR, x-ray fiber diffraction, and atomic force microscopy to reveal the 3D structure of amyloid protofilament-like fibrils formed by a 22-residue K3 peptide (Ser 20 -Lys 41 ) of ␤2-microglobulin, a protein responsible for dialysis-related amyloidosis. Although a uniformly 13 C, 15 N-labeled sample was used for the NMR measurements, we could obtain the 3D structure of the fibrils on the basis of a large number of structural constraints. The conformation of K3 fibrils was found to be a ␤-strand-loop-␤-strand with each K3 molecule stacked in a parallel and staggered manner. It is suggested that the fibrillar conformation is stabilized by intermolecular interactions, rather than by intramolecular hydrophobic packing as seen in globular proteins. Together with thermodynamic studies of the full-length protein, formation of the fibrils is likely to require side chains on the intermolecular surface to pack tightly against those of adjacent monomers. By revealing the structure of ␤2-microglobulin protofilament-like fibrils, this work represents technical progress in analyzing amyloid fibrils in general through solid-state NMR.2,2,2-trifluoroethanol ͉ amyloid fibril ͉ dialysis-related amyloidosis ͉ protein misfolding ͉ x-ray fiber diffraction

Truncated β-amyloid peptide channels provide an alternative mechanism for Alzheimer’s Disease and Down syndrome

“…Using the two available Aβ oligomer coordinate sets (27,28), we constructed annular channels based on the U-shaped β-strand-turn-β-strand motif. Previously, U-shaped motifs were also observed in the ssNMR structure of a β 2 -microglobulin fragment (29) and in the CA150 WW domain (30), and they could also form ion channels similar to prion and to β 2 -microglobulin (31,32). We constructed perfectly annular channels as the starting points for the atomistic simulations with 12 to 36 monomers per channel and lipid-favorable topology (5, 25) (SI Materials and Methods).…”

“…Early MD simulations of amyloidogenic peptides (5,25) consisting of U-shaped β-strandturn-β-strand peptides in the bilayer predicted ion-permeable channels formed by loosely attached mobile subunits with morphologies and dimensions similar to the AFM-images of amyloid channels (7,8). U-shaped motifs, first predicted by modeling of Aβ [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] (26), appear as a general feature of amyloid organization, suggesting that other U-shaped amyloid organizations may also form dynamic ion channels in the fluidic membrane (8). Because N9 and p3 have membrane-spanning segments, we modeled their 3D structures in the bilayer using the previous successful protocol (5,25).…”

Annular Structures as Intermediates in Fibril Formation of Alzheimer Aβ17−42

“…identical positions along the sequence from two neighboring peptides interacting with each other). 49,50 Comparing to β 2 -microglobulin peptide Goto and co-workers 35 have recently obtained the 3D structure of a 22-residue peptide (K3) derived from β 2 -m (residues Ser20 to Lys41). Similar to the Aβ 17-42 peptide, the K3 peptide adopts the U-shaped β-strand-turn-β-strand motif, where several inward-pointing hydrophobic residues are clustered together to form a hydrophobic core (Phe3, Phe11, Ile16, Val18, Leu20) and three negatively charged residues (Asp15, Glu17, and Asp19) at the same β-strand are pointing outward.…”

“…The structural and dynamic properties of these annularlike structures were studied in explicit water. Most interestingly, we compared the linear-like and annular-like structures of Aβ peptides with those of K3 peptides derived from the fragment of Ser20-Lys41 of the β2-microglobulin 35 , which has a similar β-strand-turn-β-strand motif in a monomeric structure. Structural comparisons suggest that amyloid structures can be dramatically different in size, structure, and morphology as a result of differences in side-chain packing arrangements, intermolecular driving forces, sequence composition, and residue positions, suggesting that the mechanism leading to distinct morphologies and the aggregation pathways is sequence specific.…”

Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR

“…From the standpoint of molecular structure, the defining feature of an amyloid fibril is the presence of cross-β supramolecular structure, meaning that the β-sheets within the fibril are formed by β-strand segments that run approximately perpendicular to the long axis of the fibril and are linked by hydrogen bonds that run approximately parallel to this axis (11)(12)(13). Although determination of the molecular structures of amyloid fibrils is made difficult by their inherent noncrystallinity and insolubility, techniques such as solid state nuclear magnetic resonance (NMR) (12,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), electron paramagnetic resonance (EPR) (34)(35)(36), electron microscopy (37)(38)(39)(40)(41)(42)(43), hydrogen/deuterium exchange (29,(44)(45)(46)(47), scanning mutagenesis (48), chemical crosslinking (27,49,50), and x-ray diffraction of amyloid-like crystals …”