Cataract-associated P23T γD-crystallin retains a native-like fold in amorphous-looking aggregates formed at physiological pH

Abstract

Cataracts cause vision loss through the large-scale aggregation of eye lens proteins as a result of ageing or congenital mutations. The development of new treatments is hindered by uncertainty about the nature of the aggregates and their mechanism of formation. We describe the structure and morphology of aggregates formed by the P23T human γD-crystallin mutant associated with congenital cataracts. At physiological pH, the protein forms aggregates that look amorphous and disordered by electron microscopy, reminiscent of the reported formation of amorphous deposits by other crystallin mutants. Surprisingly, solid-state NMR reveals that these amorphous deposits have a high degree of structural homogeneity at the atomic level and that the aggregated protein retains a native-like conformation, with no evidence for large-scale misfolding. Non-physiological destabilizing conditions used in many in vitro aggregation studies are shown to yield qualitatively different, highly misfolded amyloid-like fibrils.

Insights into protein misfolding and aggregation enabled by solid-state NMR spectroscopy

“…These kinds of non-IDP proteins form protein deposits via different types of aggregation mechanisms, with three prominent categories illustrated in Fig. 3c [32]. First, destabilization of the native fold can lead to significant unfolding that effectively opens up an amyloidogenic assembly process reminiscent of those illustrated in Fig.…”

“…However, extensive unfolding or misfolding is not a prerequisite for aggregation. Some aggregation-prone proteins self-assemble under non-denaturing conditions into non-filamentous protein deposits that lack the hallmarks of amyloids [32]. The molecular principles of these types of aggregation pathways are less well understood.…”

“…Others have advocated domain swapping as a potential contributor to this type of aggregation process, noting a correlation between domain swapping propensity and aggregation propensity. To the best of our knowledge, truly domain-swapped conformations have not yet been demonstrated within amyloid fibrils, but may be part of non-amyloid aggregates or pre-amyloid oligomers [32–35]. …”

“…This is illustrated in Fig. 3(c), in context of the condition-dependent aggregation of a cataract-related mutant eye lens protein, P23T γD crystallin[32]. Under physiological conditions this protein forms non-amyloid “amorphous” aggregates, while denaturing conditions induce formation of amyloid fibrils with a very different internal and macroscopic structure.…”

“…Indeed, many studies differentiate protein aggregates (related to disease) between elongated amyloid-like fibrils and amorphous-looking assemblies [32, 119–122]. This includes for instance pre-fibrillar oligomers formed by amyloidogenic polypeptides, which are in EM and AFM studies lacking the more ordered elongated appearance of the mature fibrils.…”