Stacked Sets of Parallel, In-register β-Strands of β2-Microglobulin in Amyloid Fibrils Revealed by Site-directed Spin Labeling and Chemical Labeling

Abstract

β2-microglobulin (β2m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β2m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β2m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β2m consist of about six β2m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.

Direct three-dimensional visualization of membrane disruption by amyloid fibrils

“…These WL fibrils are formed under different conditions to the amyloid-like fibrils of β 2 m and do not contain a highly ordered cross-β structure, as revealed by EPR, magic angle spinning NMR, and FTIR (29,30,33). Moreover, they exhibit a reduced ability to disrupt cellular function and to induce dye release from liposomes compared with their amyloid-like counterparts (27).…”

“…Previous studies have shown that these fibrils possess a parallel in register cross-β structure (29,30) assembled into multidomain filaments coiled together, described by cryo-EM (28). These fibrils bind amyloid-specific ligands such as serum amyloid P component with a similar affinity to their ex vivo counterparts (31).…”

Antiparallel β-sheet architecture in Iowa-mutant β-amyloid fibrils

“…Experimental results for Aβ 1-40 (6,8,10,11,13,14), Aβ 1-42 (5, 6, 9), Aβ 10-35 (1, 9), α-synuclein (22), amylin (20,21), β 2 -microglobulin (23,24), Ure2p (27), Sup35 (26), and PrP (28,29) fibrils support the idea that in-register parallel β-sheets might be a universal structural feature of amyloid fibrils when they are formed by full-length peptides and proteins. For HET-s fibrils, a "quasi-in-register" parallel β-sheet structure was also found, with homologous protein segments forming a parallel cross-β motif (48).…”

“…Antiparallel β-sheets with odd values of N-M occur in fibrils formed by residues 11-25 of Aβ (Aβ [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25], where the registry is 17 þ k ↔ 20 − k at pH 7.4 and 17 þ k ↔ 22 − k at pH 2.4 (31). SSNMR spectra of Aβ 11-25 fibrils show sharp lines and single sets of chemical shifts, indicating high symmetry (i.e., one molecule in the asymmetric unit).…”

“…Studies of the 40-residue and 42-residue forms of Aβ (Aβ and Aβ ) have shown that these peptides can form multiple distinct fibril structures (11,18,19), but that the cross-β motifs within wild-type (WT) Aβ fibrils are invariably comprised of in-register parallel β-sheets (1, 5, 6, 8-10, 13, 14). Parallel β-sheets have also been found in Aβ 1-40 oligomers (4) and in amyloid fibrils formed by amylin (20,21), α-synuclein (22), β 2 -microglobulin (23,24), prion proteins of yeast (25)(26)(27) and mammalian PrP (28,29); in contrast, antiparallel β-sheets have been found in fibrils formed by Aβ fragments with 15 or fewer residues (30)(31)(32) and in amyloid-like crystals of certain Aβ fragments (33). These observations suggest that in-register parallel β-sheets might be a universal feature of amyloid structures in which the polypeptide chain contains more than one β-strand-forming segment.…”