Release of frustration drives corneal amyloid disaggregation by brain chaperone

Abstract

TGFBI-related corneal dystrophy (CD) is characterized by the accumulation of insoluble protein deposits in the corneal tissues, eventually leading to progressive corneal opacity. Here we show that ATP-independent amyloid-β chaperone L-PGDS can effectively disaggregate corneal amyloids in surgically excised human cornea of TGFBI-CD patients and release trapped amyloid hallmark proteins. Since the mechanism of amyloid disassembly by ATP-independent chaperones is unknown, we reconstructed atomic models of the amyloids self-assembled from TGFBIp-derived peptides and their complex with L-PGDS using cryo-EM and NMR. We show that L-PGDS specifically recognizes structurally frustrated regions in the amyloids and releases those frustrations. The released free energy increases the chaperone's binding affinity to amyloids, resulting in local restructuring and breakage of amyloids to protofibrils. Our mechanistic model provides insights into the alternative source of energy utilized by ATP-independent disaggregases and highlights the possibility of using these chaperones as treatment strategies for different types of amyloid-related diseases.

Fig. 1. Atomic model of the G623R fibril reconstructed using ssNMR and cryo-EM.

a 2D NCA and (b) 2D NCO (Contour level: 7e + 006, 37, 1.1) (c) 2D ¹³C-¹³C DARR with contact time 20 ms and 75 ms spectra of the ¹³C-¹⁵N labeled G623R fibril represented in red and orange, respectively. Residues coming from the extended conformation and β turn regions are colored in blue and green, respectively. (Contour level: 1.5e + 006, 37, 1.1). d Representative cryo-EM image of the G623R fibril. e 2D class average of the fibril and its respective power spectrum showing distance between meridian line and 1^st layer line of 4.75 Å. f Calculated model of G623R fibril comprising 5 repeating units each consisting of 4 monomers.

Fig. 2. Binding of L-PGDS to the G623R fibril.

a Representative cryo-EM image of the G623R fibril and L-PGDS conjugated with heme for contrast enhancement. The red arrow shows the localization of L-PGDS along the convex surface of the fibril. b Snapshot of MD simulation of the G623R fibril (light gray) and L-PGDS (olive green) complex at time step 6 ns before the opening of the salt bridge (E615 and R623). The blue and black arrows represent the normal of the plane of 2nd and 9th monomer defined by CA of A620, N622 and H626, respectively. The angle α is formed between the normal of the 2^nd and 9^th planes. The interacting monomer is highlighted in red.

Fig. 3. Mapping of interactions between G623R fibril and L-PGDS.

a Selected residues of ¹H-¹⁵N HSQC spectra of the ¹⁵N-labeled L-PGDS in the absence (blue) and presence of G623R fibrils (red). b ¹³C-¹³C DARR spectra of the ¹³C and ¹⁵N-labeled G623R fibrils in the absence (green) and presence of L-PGDS (orange). c, d Residues of L-PGDS and G623R fibril, respectively, exhibiting significant chemical shift changes as highlighted in red and yellow. e NMR data-guided docking model of G623R fibril and L-PGDS (PDB code: 4IMN).

Fig. 4. Interaction interface between L-PGDS and the G623R fibril.

a Zoomed-in image of the docking model displaying electrostatic interaction between E615, D617 of G623R fibril and K58, K59 & R85, K86 of L-PGDS, respectively. b End point ThT assay showing the ThT fluorescence intensity of the G623R and D619K fibril, its complex with L-PGDS, mutant K58D of L-PGDS, and mutant K58A, K58A of L-PGDS (ΔL-PGDS) n  ≥  3 independent experiments. c Zoomed-in 3D representations of the interactions between G623R with mutant K58D of L-PGDS, mutant D617K of G623R with L-PGDS, mutant D617K of G623R with mutant K58D of L-PGDS. The negatively charged ASP residue is colored in red and the positively charged LYS residue is colored in blue. d Histogram showing the end-to-end distance of G623R fibril incubated with PBS buffer only, L-PGDS and double mutant (K58R, C65A) of L-PGDS (RA L-PGDS).

Fig. 5. Ex vivo disaggregation effects of L-PGDS on corneal samples.

Corneal images collected from a patient with R124C mutation with Congo red staining incubated with (a) control PBS buffer and (b) the same buffer containing 10 μM L-PGDS. The amyloid deposits are indicated by the black arrow. The histogram shows the distribution of the area of amyloid deposits in corneal samples treated PBS control and 10 μM L-PGDS.