Figure 1. The number of α-Syn monomers/oligomer at different times
during formation. Most oligomers have less than 12 monomers in studies
by Cremades et al. [2] (copied with permission). We added dashed
lines to indicate 6, 12, 18, and 30 monomers: the number of monomers in
some of our models.
Even the secondary structures of the assemblies alter with time.
Recently Zhou and Kurouski [3] used atomic force microscopy-infrared
spectroscopy to analyze the structure of α-Syn oligomers at different
stages of aggregation. The nanometer resolution of this approach allows
different aggregate species to be analyzed simultaneously. They observed
high heterogeneity. Initially most assemblies were dominated by random
coil and α-helical structures, but some contained β-sheets (mostly
antiparallel). With time the amount of random coil/α-helical and
antiparallel β-structure decreased while that of parallel β-structures
increased. By day 7 parallel β-fibrils dominated, but some spherical
assemblies with significant antiparallel β-content persisted. Lashuel et
al. [4] obtained electron microscopy (EM) images of α-Syn annular
protofibrils that assemble to form tubular protofibrils that then morph
into fibrils. Chen et al. [5] developed cryoEM images of toxic
cylindrical oligomers composed of ~ 18 and 29 α-Syn
monomers. Fibrils are the only assemblies for which atomic-scale
structures have been determined, and then only for portions of α-Syn
[6-11]. They all include at least some segments from residues 15 –
97 that form in-register parallel cross-beta sheets. However, the exact
sequential locations of the β-strands, overall folding patterns of the
monomers, and interactions between fibrils vary among these structures.
Also, these fibrils may not be the same as those occurring in the human
brain [12]. None appear consistent with previously reported EM
images of ribbons [13,14].
But functional roles of synucleins and their pathologies may involve
interactions with lipids and/or fatty acids absent in all thein-vitro studies mentioned above (reviewed in Mori et al.
[15]). The first 95 residues of α-Syn form two antiparallel helices
on the surface of negatively charged membranes [16]. These
interactions are especially prevalent in presynaptic vesicles where
α-Syn apparently interacts with other proteins. However, Synucleins also
occur elsewhere and aggregate into larger assemblies. Lewy bodies
commonly found in PD patients contain large quantities of synucleins and
amyloid beta amyloids but are also chocked full of lipids, fatty acids
and organelles. Meade et al. [1] observed a series of helical α-Syn
polymorphs that occur in the presence of lipid vesicles.
Giorgia De Franceschi et al.
[17] observed similar fibrils when the ratio of docosahexaenoic acid
to α-Syn is relatively low. These lipid-α-Syn fibrils have much larger
diameters than those formed in the absence of such amphiphiles. The
α-Syn conformation is dependent upon the ratio of lipid to α-Syn; when
the ratio is high α-helical oligomers form on the surface of lipid
droplets [17]. In contrast, van Diggelen et al. [18] identified
two α-Syn/fatty acid oligomer assemblies that were both rich in
antiparallel β-structure but had differing epitopes. Eichmann et al.
[19] have published EM images of high-density lipoproteins formed by
all three families of synucleins in the presence of sphingomyelin. Also,
numerous studies have found that α-Syn permeabilizes membranes and forms
channels in both artificial membranes [20-23], and neural cells
[24-26].
Several familial mutations that contribute to PD have been identified,
most of which are sequentially near each other. However, it is unclear
how or whether these alter oligomer conformations or the binding of this
segment into a cleft within the Cyclophilin A protein [27].
Findings that numerous α-Syn assemblies typically coexist in
equilibrium, alter slowly with time, have only partially ordered and/or
fractionally occupied secondary structures, and are affected by
cofactors, complicates experimental determination of their structures
and what makes some toxic. Also, computational modeling that relies on
molecular dynamic simulations of randomized assemblies is problematic
because oligomeric and fibril development often occurs over days or
weeks and involves many monomers whereas most molecular dynamic
simulations last less than a millisecond for a relatively small number
of monomers. Furthermore, assembly conformations may depend upon factors
not included in the simulations.
We utilize an alternative approach to modeling oligomers and
transmembrane channels that concentrates on β-barrels, most of which are
concentric. Previously we published concentric β-barrel models of
amyloid-β42 (Aβ42) hexamers, dodecamers, annular protofibrils, and
transmembrane channels [28-30]. Several aspects of our models
including six-stranded antiparallel β-barrels and concentric β-barrels
were unprecedented. Since then, however, determination of the structures
of two channel-forming toxins, lysenin [31] and aerolysin [32],
have demonstrated that assemblies composed of multiple identical
subunits related by radial symmetry and that contain concentric
β-barrels occur naturally. Also, Laganowsky et al. [33] found that a
segment from an amyloid-forming protein, alphaB crystalline, can adopt a
radially symmetric six-stranded antiparallel β-barrel hexamer structure
(which they call cylindrin) similar to the core β-barrel of our Aβ42
hexamer model, Do et al. [34] found that the sequences of portions
of Aβ can also form this motif, and Serra-Batiste et al. [35] found
that Aβ42 channels have β-barrel structures with only two distinct
monomer conformations.
Stimulated by these and other supportive experimental findings, we have
renewed our modeling efforts. Although some details of our current
generation of models have been altered and are still evolving, the basic
concept of assemblies composed of concentric β-barrels [30] has been
retained. Here we extend those concepts and techniques to develop models
of a variety of non-fibril Synuclein assemblies. Given the difficulties
described above, attempts to model these assemblies in the absence of
high-resolution structural data may seem ludicrous. A saving grace is
that numerous lower resolution microscopy studies have revealed circular
and cylindrical assemblies composed primarily of β secondary structure.
The simplest way to model circular and cylindirical β-structures is as
β-barrels, which are highly constrained if all subunits are identical
and they are symmetric and concentric.