Isomerization of L-Aspartate (L-Asp) into D-aspartate (D-Asp) is a spontaneous non-enzymatic reaction that has been used in molecular dating of tissue samples across a wide range of age estimations1, 2 (Figure 1a). Accumulation of D-Asp in lens crystallin is found to be age-dependent, and the racemic ratio of D/L-Asp correlates well with aging. D-Asp has been since identified in several misfolding disease-associated proteins, including tau, myelin basic proteins, and amyloid beta (Aβ) peptide 1–42 (Aβ42).3, 4, 5, 6, 7, 8 Importantly, D-Asp has been found in Aβ42 aggregates at positions 1 and 79 and tau protein aggregates4 in the brains of Alzheimer’s disease (AD) patients. Aβ42 has three Asp residues at positions 1, 7, and 23. They are located near the hot spots for familial Alzheimer's disease (FAD) mutations and phosphorylation sites of serine (Ser) 8 and 26, indicating that structural alterations in these hotspots may increase the aggregation and neurotoxicity of Aβ42 (Figure 1b).10 Indeed, in vitro aggregation kinetics monitored by thioflavin T (ThT) shows that the replacement of naturally occurring L-Asp with D-Asp by chemical synthesis can accelerate the kinetics of amyloid fibril formation of Aβ4211, fragments of tau3 and amylin.12 Additionally, the introduction of isoaspartyl residue (isoAsp), an intermediate of the L- to D-Asp isomerization also found in AD patients (Figure 1a), to the residue position 23 of Aβ42 can lead to increased aggregation propensity and neurotoxicity13. Such a modification has indeed been identified in patient tissues.14 At a molecular level, D-Asp-containing Aβ peptide 20–29 adopts a significant amount of polyproline II (PPII) type structure as opposed to a random coil-like structure for the L-Asp-containing counterpart.15 Introduction of D-Asp also alters the morphology of amyloid fibrils of the R2 and R3 fragments of tau.16

Neurodegenerative diseases are closely associated with amyloid fibril formation of Aβ, tau, α-synuclein, and other intrinsically disordered proteins17, 18, 19. These protein aggregates are highly polymorphic, and their assemblies are acutely dependent on the experimental conditions under which the amyloid fibrils are generated20, 21, 22, 23. Recent advances in cryo-electron microscopy (cryo-EM) have generated a library of atomic structures of amyloid fibrils extracted directly from postmortem human brains.24, 25, 26, 27, 28 These clinical amyloid fibril structures exhibit distinct structural features compared to those prepared in test tubes. These brain-derived amyloid fibrils shed light on the polymorphism pertinent to different disease backgrounds. However, the effect of racemization of aspartates on amyloid fibril formation has not been characterized in atomic details to date. To address this issue, we chemically synthesized two Aβ42 peptides harboring D-Asp at position 23 (Aβ42 D-Asp23) and at positions 7 and 23 (Aβ42 D-Asp7,23) to examine the effect of aspartate isomerization on the aggregation kinetics and amyloid polymorphism of Aβ42. We found that Aβ42 D-Asp23 and Aβ42 D-Asp7,23 exhibit increased and reduced lag phase of aggregation kinetics compared to that of wild type (WT). Cryo-EM analysis showed that Aβ42 D-Asp23 and Aβ42 D-Asp7,23 share a similar double-stranded amyloid fibril structure, while Aβ42 D-Asp7,23 exhibits an additional triple-stranded amyloid fibril structure. The double- and triple-stranded assemblies observed in this study are unique among all known Aβ amyloid fibril structures reported in the literature to date. Our findings illustrated how the racemization of aspartates in Aβ42 peptides may sporadically impact neurodegeneration.