Structural polymorphism of ex-vivo ALECT2 amyloid fibrils revealed by cryo-EM

Abstract

ALECT2 amyloidosis is a rare systemic disease characterized by the pathological deposition of leukocyte cell-derived chemotaxin-2 (LECT2) as amyloid fibrils, primarily affecting the kidneys and liver. The molecular mechanisms underlying LECT2 aggregation remain poorly defined, hindering diagnostic and therapeutic development. Here, we present cryo-electron microscopy structures of ex-vivo ALECT2 fibrils extracted from a patient's kidney. We identified three fibril polymorphs: a predominant single-protofilament morphology and two minor double-protofilament morphologies. The dominant single-protofilament morphology comprises the full-length 133-residue LECT2 protein and retains all three native disulfide bonds. Low-resolution reconstructions of double-protofilament morphologies suggest they adopt a similar fold to the single protofilament morphology, but form paired assemblies with different inter-filament interfaces. Mass spectrometry also reveals acetylation within the fibrils. These findings offer critical insights into the structural basis of ALECT2 amyloid formation and identify molecular features that could inform future diagnostic and therapeutic approaches.

Fig. 1.. Characterization of ALECT2 amyloid deposits in kidney.

a. Histological characterization of kidney sections using (left to right) H&E stain, Congo red, and Thioflavin-S staining. Scale bar, 50 μm. b. Negative stain electron microscopy image of fibrils extracted from the kidney. Scale bar, 200 nm. c. Proteomic analysis using tryptic (red solid line) and chymotryptic (CT, blue solid line) digestion highlights peptide coverage by mass spectrometry. Cysteines are colored pink and the Val40 is colored green.

Fig. 2.. Cryo-EM analysis of ALECT2 fibrils.

a. Representative cryo-EM micrographs showing single protofilament morphology (left) and double protofilament morphology (right). Scale bar: 20 nm. b. Representative 3D averages of ALECT2 fibrils. The left 3D class corresponds to the single-protofilament morphology, while the 3D classes on the right represent two distinct double-protofilament morphologies, double protofilament 1 and double protofilament 2 respectively. c. Single layer of ALECT2 fibril cryo-EM density map and its built-in atomic model. All 133 residues of the mature LECT2 protein are incorporated into the fibril structure, with their disulfide bonds retained (colored in yellow) and Val40 in gray dotted square. Extra densities that are predicted to be non-proteinaceous are drawn in black. d. Sequence of LECT2: scheme of the secondary structure elements of the single protofilament morphology. β-strands are numbered and marked with green arrows.

Fig. 3.. Stabilizing interactions within the ALECT2 single protofilament morphology.

a. The top view of the fibril highlights stabilizing interactions in red; disulfide bonds are shown in yellow. The N-terminal region of the fibril, spanning residues Gly1 to Gly45, is shaded in light pink color, while the C-terminal region, encompassing residues Ser46 to Leu133, is shaded in light blue. b. Side view of five layers reveals an angular offset between the N- and C-terminal regions of ~7°. c-g. Detailed views of examples of stabilizing interactions. c. Hydrogen bond between Tyr132 (n+2) and Tyr92 (n). d. Intra-layer hydrogen bond between Gln29 and Ser27. e. Salt bridge between Lys97 and Glu100 within the same layer. f. Salt bridge between Arg16 (n+1) and Glu14 (n). g. Salt bridge between Asp19 (n+1) and Arg20 (n). These interactions collectively contribute to the structural integrity of the fibril.

Fig. 4.. Cryo-EM reconstruction of the minor double protofilament morphologies.

Cryo-EM map and docked model of the a. double protofilament 1 (top) double protofilament 2 (bottom) morphologies. The model for the single filament morphology was used to dock each of the two double protofilaments. b. Panels highlights the interface formed between the protofilaments in both morphologies. Scale bar, 3Å. c. Upper and lower panels are representative cryo-EM micrographs tracing the single protofilament (green), double protofilament 1 (blue) and double protofilament 2 (orange). Scale bar, 20 nm. Twenty individual micrographs were used; the representing figure is from two micrographs.

Fig. 5.. APR prediction in LECT2.

a. Heat maps of aggregation-prone regions (APR) mapped onto the LECT2 native structure (PDB ID: 5B0H, left) and the ALECT2 single protofilament structure (PDB ID: 9NON, right) and, with scores ranging from 0 to 4 (gray to dark rust), where 0 indicates the lowest likelihood of aggregation and 4 represents the highest. b. ALECT2 sequence with secondary structure annotations for both the native and fibril forms, with APRs color-coded according to a.