Stabilizing Aβ globulomers: the Aβ(L17C/L34C) hairpin
Globulomers are, as mentioned, spherical Aβ oligomers that form in SDS- or fatty acid-containing solutions. Initially, in 0.2% SDS, smaller 16 kDa aggregates, referred to as preglobulomers, form and these associate into the 64 kDa globulomers upon dilution of the SDS content. The solution NMR studies, carried out by Yu et al. , of preglobulomers formed by Aβ42 (with an N-terminal methionine) are the most detailed structural characterization reported so far of any soluble oligomeric form of wild-type Aβ. Several resonances in the NMR spectrum of preglobulomers were resolved and could be assigned. Different isotopic labeling and peptide-mixing schemes then allowed the authors to distinguish between interchain and intrachain NOE connectivities. NOE and chemical shift data were used to determine the structures of dimeric Aβ units within the preglobulomer. In these, residues 18-23 and 28-33 formed an intramolecular two-stranded antiparallel β-sheet connected by a turn involving residues 24-27. The dimer was then formed by intermolecular interactions by the two 34-39 fragments in a two-stranded parallel β-sheet.
Based on this conformation, Yu et al. introduced an L17C/L34C double mutation to stabilize the hairpin with an intramolecular disulfide. The L17C/L34C mutant forms stable and homogenous globulomers that are recognized by a conformation-selective monoclonal antibody towards wild-type globulomers . These stable globulomers can be used for further structural and functional studies.
It is very interesting to note that the intrachain NOE connectivities which are observed in NMR spectra of the preglobulomer  are consistent with the hairpin conformation of Aβ that we previously observed in complex with the ZAβ3 Affibody [40,41] (Fig. 1A). Monomeric Aβ does not adopt a unique conformation in an aqueous solution . Nevertheless, NMR experiments and molecular modeling indeed suggest that Aβ has a propensity to form ‘β-hairpin’ conformations in which the nonpolar fragments 17–21 and 31–36 are extended strands connected by a turn in the region 24–28 [42–45]. The observation of precisely such structures of Aβ in globulomers and in complex with a binding protein therefore adds considerable support to the notion that the β-hairpin is an accessible, and even pervasive, structure of nonfibrillar Aβ.
Figure 1. Protein engineering to stabilize oligomeric aggregates of Aβ and avoid aggregation into insoluble amyloid fibrils. (A) The β-hairpin conformation of Aβ40 observed in complex with an Affibody binding protein . Nonpolar side chains at the two hydrophobic faces are shown as sticks and colored yellow and orange, respectively. The Ala21 and Ala30 methyl groups are located in close proximity on opposite β-strands. (B) Schematic of an Aβ aggregation mechanism, which involves the β-hairpin as a transient conformation sampled by the monomer and as a constituent of oligomeric Aβ . Soluble oligomers thus contain antiparallel β-sheet secondary structure and a conformational transition is required to create a fibril seed with in-register, parallel β-sheets. Orange side chains form the core in the cross-β conformation of the fibril seed . The mechanism was initially suggested based on the observation of the β-hairpin conformation of monomeric Aβ  and FTIR data of Aβ oligomers , which indicate antiparallel secondary structure content. The oligomer secondary structure was confirmed  and a β-hairpin conformation as in (A) was subsequently observed in globular Aβ oligomers . (C) The AβA21C/A30C double mutant (Aβcc) in which the β-hairpin conformation is locked by a disulfide bond. Aβcc forms soluble oligomers and protofibrils, but not amyloid fibrils, as depicted in (B). A similar strategy was used to stabilize globulomer Aβ aggregates .
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It is therefore also not inconceivable that the disulfide in the L17C/L34C double mutant results in a structure which is similar to that of the A21C/A34C double mutant (Aβcc; described below), which was engineered based on the structure of Affibody-bound Aβ . This is because the two double mutations are expected to lock the two-stranded antiparallel β-sheet of the hairpin into the same register. The distance between the two β-carbons of L17 and L34 in Aβ in the Affibody complex is, on the other hand, almost 6 Å (i.e. the L17C/L34C disulfide engineered by Yu et al.  might not be completely compatible with the hairpin conformation that is stabilized in Aβcc ).
The β-hairpin structure is topologically very similar to the conformation of Aβ in amyloid fibrils with a ‘cross-β’ structure [46–48]. However, there is a distinct difference in that hydrogen bonds in the hairpin are intramolecular, resulting in antiparallel β-strands, whereas they are intermolecular in fibrils, resulting instead in parallel β-sheets. Other studies also demonstrate the importance of a reverse turn at the center of the 23–28 fragment in aggregates of Aβ. For instance, a lactam bridge linking the side chains of D23 and K28 in Aβ40 increases the rate of amyloid fibril formation by a factor of ∼ 1000 .
Aβ(A21C/A30C) hairpin (Aβcc)
The kinetics of in vitro fibril formation involves a lag time, a nucleation (seeding) event and a runaway fibril-formation reaction. The aggregation is also enhanced by breakage of fibrils to form new seeds [50,51]. A comparison of structural features of monomeric Aβ and the conformation of Aβ in fibrils led us to suggest that the aggregation of Aβ involves oligomeric intermediates composed of β-hairpins that undergo a concerted conformational change, much like closing Venetian blinds, to form fibril seeds  (Fig. 1B). To test the mechanism, we sought ways to allow Aβ to form β-hairpins while restricting a conformational change into the cross-β conformation; if the hypothesis was correct, this should allow the enrichment of stable soluble oligomeric species. Examination of the β-hairpin structure observed in the Affibody complex revealed that the β-carbons of alanine 21 and alanine 30 are located in close proximity to each other on opposing strands and that a disulfide would be accommodated without disrupting any other conformational properties of the hairpin, such as backbone hydrogen bonding (Fig. 1C). The disulfide would, on the other hand, not be compatible with the cross-β structure observed in fibrils. Hence, we created the Aβcc [Aβ(A21C/A30C)] double mutant . Aβcc may potentially also form intermolecular disulfides. However, the production system , in which Aβ (with an N-terminal methionine) is obtained as an Aβ–Affibody complex, allows for purification of monomeric Aβcc with an intramolecular disulfide.
We find that Aβcc indeed readily forms soluble oligomeric and protofibrillar species but not amyloid fibrils, unless the restraining disulfide is broken by reduction. Briefly, Aβcc can form two types of oligomeric species that aggregate further into either protofibrils that are recognized by the mAb158 monoclonal antibody, which is selective for protofibrils of wild-type Aβ , or into amorphous aggregates that are recognized by the (polyclonal) A11 antibody . (There is not much cross-reactivity between the A11 and mAb158 antibodies.) We have named these two aggregation pathways based on secondary structure features of the originating oligomers. Oligomers with an apparent molecular mass of ∼ 100 kDa (as observed in size-exclusion chromatography) that contain antiparallel β-sheet secondary structure (as indicated by CD and infrared spectroscopy) aggregate along the ‘β-sheet pathway’ to form protofibrils that are detectable by mAb158. Smaller oligomers, without regular secondary structure, aggregate along the ‘coil pathway’ to form A11-binding species. (The resulting secondary structure within the A11-binding aggregates is not yet known.) There is also a cross-over option available to smaller (‘coil’) oligomers: they can form β-sheet oligomers and ultimately protofibrils when concentrated to millimolar peptide concentrations.
The 100-kDa β-sheet oligomers and/or protofibrils formed by these on the β-sheet aggregation pathway induce apoptosis in SH-SY5Y (a human-derived cell line that was subcloned three times from a bone marrow biopsy of a metastatic neuroblastoma site of a young woman)  neuroblastoma cell lines . The toxicity is comparable to, or larger than, that of wild-type Aβ oligomers, prepared as described previously . Aβ42cc forms toxic species more readily than Aβ40cc. This is because the longer peptide preferentially aggregates along the β-sheet pathway – oligomers of Aβ40 that have been forced to cross over into the β-sheet pathway in concentrated samples also induce apoptosis.
The fact that Aβcc aggregates via two distinct pathways is consistent with studies of wild-type Aβ aggregation [26,57,58]. The size and secondary structure content of β-sheet oligomers formed by Aβcc are reminiscent of those of globulomers. The β-sheet oligomers also share the non-A11 binding and apoptotic properties of naturally occurring ASPDs, described above [26,27].
Protofibrils are the most stable form of oligomeric Aβcc. TEM images reveal that these are morphologically very similar, if not identical, to protofibrils of wild-type Aβ. They are ∼ 6 nm in diameter and up to 60 nm in length and appear as curved (or flexible) beaded strings in which individual beads are spherical. It is not unlikely that the 6-nm beads correspond to the original β-sheet containing oligomers that formed the protofibrils, but changes in secondary structure content upon fibril formation cannot be completely ruled out. However, Fourier transform infrared (FTIR) spectroscopy of protofibril samples indicates that antiparallel β-sheet structure is present in these. Presumably this originates from the (restricted) hairpin conformation of Aβcc.
As mentioned, samples of soluble Aβ isolated from the brains of AD patients contain small oligomers, primarily dimers, which do not dissociate in SDS/PAGE [22,24]. Small amounts of SDS-resistant oligomers also form in in vitro preparations of wild-type Aβ42. It is interesting to note that Aβcc forms such SDS-resistant dimers and trimers more readily than wild-type Aβ. In particular, Aβ42cc species from the β-sheet oligomers only appear as dimers and trimers (no monomers) in SDS/PAGE . Still, further research is needed to determine if they also share other biochemical and structural properties with the AD dimers.