Mechanisms of Protein Oligomerization: Inhibitor of Functional Amyloids Templates α-Synuclein Fibrillation

Abstract

Small organic molecules that inhibit functional bacterial amyloid fibers, curli, are promising new antibiotics. Here we investigated the mechanism by which the ring-fused 2-pyridone FN075 inhibits fibrillation of the curli protein CsgA. Using a variety of biophysical techniques, we found that FN075 promotes CsgA to form off-pathway, non-amyloidogenic oligomeric species. In light of the generic properties of amyloids, we tested whether FN075 would also affect the fibrillation reaction of human α-synuclein, an amyloid-forming protein involved in Parkinson’s disease. Surprisingly, FN075 stimulates α-synuclein amyloid fiber formation as measured by thioflavin T emission, electron microscopy (EM), and atomic force microscopy (AFM). NMR data on 15N-labeled α-synuclein show that upon FN075 addition, α-synuclein oligomers with 7 nm radius form in which the C-terminal 40 residues remain disordered and solvent exposed. The polypeptides in these oligomers contain β-like secondary structure, and the oligomers are detectable by AFM, EM, and size-exclusion chromatography (SEC). Taken together, FN075 triggers oligomer formation of both proteins: in the case of CsgA, the oligomers do not proceed to fibers, whereas for α-synuclein, the oligomers are poised to rapidly form fibers. We conclude that there is a fine balance between small-molecule inhibition and templation that depends on protein chemistry.

Targeting α-Synuclein as a Parkinson’s Disease Therapeutic

“…For example, it is reported that in vivo, α-synuclein filaments are 14-16 nm thick [129] and have lengths !1 μM. In contrast, on-pathway (to fibril formation) oligomers are reported to have a Stokes hydrodynamic radius of 7 nm [130,131] and are much shorter. Spherical oligomers, which may be among the toxic species [107], are even smaller at 2-6 nm in diameter, whereas annular oligomers appear under AFM as a dense, remarkably homogeneous population of globular, mildly flattened oligomers with a narrow size distribution (average diameter of 21 AE 3 nm) and with a less dense interior or a pore-like toroid [106,112].…”

“…Conversely, a particular molecule may have opposing effects on structurally related targets, for example, by promoting off-pathway oligomers of one target but promoting fibrillization of another [131]. Thus, structure-based predictions regarding mechanism of binding and resulting α-synuclein species are difficult.…”

Curli biogenesis: Order out of disorder

“…A screen of compounds originally designed to inhibit the elaboration of E. coli P pili yielded a set of small molecules capable of inhibiting amyloid formation of Aβ [99, 100]. These compounds have since been subject to extensive chemical modification and assayed for biological activity against several amyloid substrates including curli [28, 91, 101]. This extensive library of 2-pyridone compounds, or curlicides, exhibit varying degrees of CsgA amyloid inhibition and curli-dependent biofilm inhibition [28, 91, 101].…”

Structural, morphological, and functional diversity of amyloid oligomers

“…Analytical ultracentrifugation (AUC) measurements combined with the H/D exchange [41,42] and fluorescence analyses [43] have shown that amyloid core (residues 40-80) is consistently protected from the solvent in oligomers, and in some oligomer preparations this protection is extended on all N-terminal half of the protein (residues 1-90) [41]. Oligomers with smaller solvent-protected core were shown to be not on the pathway to fibril formation, whereas the oligomers with larger protected core were easily converted to fibrils [41,44].…”