Fig. 3: Overall structure of the hnRNPDL-2 fibril core.

a Schematic representation of one cross-sectional layer of the hnRNPDL-2 fibril core. The ___location of the β-strands is indicated with thicker arrows. Polar and hydrophobic residues are colored in green and white, respectively. Glycine residues are colored in yellow. Aspartic residues are colored in red. b Sequence alignment of hnRNPDL orthologues showing evolutionary conservation of residues within exon 6. The β-strands that build the hnRNPDL-2 fibrils amyloid core are indicated. The mutated D259 and surrounding conserved residues are shown in red and blue, respectively. Conserved residues are indicated with an asterisk. c Surface representation showing the electrostatic surface potential of the hnRNPDL-2 fibril at pH 7, with ribbon representation of one subunit on top. Electrostatic potential maps were calculated using APBS server and visualized using the APBS plugin in PyMol (Schrödinger, NY, USA)^52,59. Negative and positive map potential values are colored in red and blue, respectively, according to the k_bTe_c⁻¹ unit scale (k_b is the Boltzmann’s constant, e_c is the charge of an electron and a T is 298 K). The ___location of the six β-strands (β1 to β6) is indicated. The side chain of D259 is shown as blue sticks and labeled. d Stick model of one hnRNPDL-2 fibril rung showing the position of evolutionary conserved residues (colored in pink). D259 disease-associated residue is highlighted in red. e Upper panel, top view of the Y255 to Y260 segment showing the interlayer interactions between Y260 and G256. Lower panel, close-up view perpendicular to the fibril axis showing the Y260 packing and their interactions with the main chain of G256. Y260 side chains are shown as sticks (colored in cyan) over the cryo-EM map sown as a grey mesh. f Effect of LGMDD3-associated mutations on hnRNPDL-2 fibril formation. The WT hnRNPDL-2 and D259H and D259N mutant proteins were incubated under the same conditions and subjected to separation on a glycerol cushion and SDS–PAGE analysis (fractions from top to bottom, 1–6). Bottom panels, representative NS-TEM images of amyloid filaments from the bottom fraction. This experiment has been performed twice with similar results.