Fig. 4: Structure of the RNA/DNA-binding domains from hnRNPDL-2 and their ___location in the amyloid filaments. | Nature Communications

Fig. 4: Structure of the RNA/DNA-binding domains from hnRNPDL-2 and their ___location in the amyloid filaments.

From: Cryo-EM structure of hnRNPDL-2 fibrils, a functional amyloid associated with limb-girdle muscular dystrophy D3

Fig. 4

a Top panel, representative 2D class average image showing hnRNPDL-2 fibrils with an approximate solenoid coat of globular domains. The arrows indicate the ___location of the density assigned to the RRMs. Bottom panel, negative-stain electron microscopy (EM) micrographs of hnRNPDL-2 filaments bound to nanogold-antibodies (~10 nm, white arrow) targeting the ___location of the hnRNPDL-2 globular domains. The N-terminal 6xHis tag was labelled using nanogold-conjugated secondary antibodies against anti-6xHis antibodies produced in mouse. Representative image from two independent experiments. Scale bar, 50 nm. b 3D unsharpened density map reconstruction of hnRNPDL-2 fibril. Densities for the amyloid core and putative RRMs are colored in blue and pink, respectively. The 10x filament rise/subunit is indicated in Å. c Schematic diagram showing the proposed organization of the RRM domains (pink) around the fibril core (cyan). d Cross-sectional view of the unsharpened cryo-EM map of hnRNPDL-2 fibril, with the superimposed hnRNPDL-2 amyloid core in a ribbon representation. The side chain of F231 is represented as sticks. e Model structure of the N-terminal RNA-binding domains RRM1 and RRM2 from hnRNPDL-2 generated with AlphaFold260. f Electrophoretic mobility shift assay (EMSA) of soluble hnRNPDL-2 with a Fluorescein-labelled oligonucleotide (F-ssDNA). The 7-mer ssDNA was incubated with the soluble form of hnRNPDL-2 at the indicated protein concentrations. EMSA has been performed three times with similar results. g Binding affinity of soluble hnRNPDL-2 to the 7-mer fluorescent ssDNA (F-ssDNA) determined by the EMSA assay. Data is shown as mean ± SEM (n = 3 independent experiments). h Binding of the 7-mer fluorescent ssDNA (F-ssDNA) to preformed hnRNPDL-2 amyloid filaments. Data is shown as mean ± SEM (n = 4 independent experiments for all protein concentrations, except for 10 and 25 µM with n = 3 independent experiments). i Representative confocal microscopy image of hnRNPDL-2 amyloid fibrils bound to fluorescent ssDNA (+F-ssDNA). Control fibrils without F-ssDNA are shown as control condition. Representative image from three independent experiments. In (g) and (h) data was fitted to one-site specific binding mechanism with Hill slope using GraphPad Prism57.

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