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. 2021 Sep 7;10:e67605. doi: 10.7554/eLife.67605

Figure 5. Key residues governing the RRM-dependent TDP-43 multimerization on RNA targets.

(a) Superimposition of calculated (red curve) and experimental (black dots) SAXS curves corresponding to RRM1–2 bound to (GU)12. SAXS curves were calculated from all-atoms model using the program GAJOE from the suite EOM. The corresponding χ2 values are indicated. The inset is a 3D representation of the model built using MD simulations from which the conformational state at equilibrium was considered. (b) Zoom in on the 3D model corresponding to the RRM1–2 bound to (GU)12 showing the protein-protein interface created by the interaction of residues located in the α -helix α1 and β-strand β2 belonging to the RRM2 (first monomer) with residues located in the RRM1 loop 3 (second monomer). Interacting couples are highlighted in red and interaction bonds are shown by dotted lines. Numbers in black reflect the distance (Å). (c) Upper panel shows a zoom in on the superimposed 1H-15N SEA-HSQC spectra (see full NMR spectra in Figure 5—figure supplement 5) of 15N-labeled RRM1–2 bound to (GU)6 (turquoise) or to (GU)12 (magenta). The residues present at RRM1–2 dimerization interface (highlighted in red) are no longer exposed to the solvent. Lower panel shows the global changes derived from SEA-experiment in solvent-exposed amides located at the protein-protein interface which are mapped on the 3D structure obtained from MD simulations (blue: exposed, red: not exposed). (d) As in Figure 3c, the microtubule bench assay was used to quantify the compartmentalization of different forms of TDP-43 co-expressed in HeLa cells. Center panel: view on the close proximity between R208 in RRM2 (first monomer) and K137 in RRM1 (second monomer). Upper panel shows a demixing phenotype between wild-type and K137E TDP-43. In contrast, R208 better mixes with K137E than wild-type TDP-43. Bottom panel, as a control, this behavior is not observed in the case of K140E. Scale bar: 10 µm. p<0.05*; p<0.01** (paired two-sample Kolmogorov–Smirnov test). n.s. non-significant. N, number of compartments.

Figure 5—source data 1. Small-angle X-ray scattering (SAXS) data of RRM1–2 bound to (GU)12 (see legend of Figure 5a).
Figure 5—source data 2. The microtubule bench assay was used to quantify the compartmentalization of different forms of TDP-43 co-expressed in HeLa cells (See legend of Figure 5d).

Figure 5.

Figure 5—figure supplement 1. Small-angle X-ray scattering (SAXS) analysis of TDP-43 RRM1–2 fragment alone or bound to GU-repeats.

Figure 5—figure supplement 1.

(a) Superimposition of calculated (red curve) and experimental (black dots) SAXS curves for the free RRM1–2 (top left panel), RRM1–2 bound to (GU)3 (bottom left panel), to (GU)6 (top right panel), and to (GU)12 (bottom right panel). SAXS curves were calculated from all-atoms models using the program GAJOE from the suite EOM. The corresponding χ2 values are indicated. The insets are 3D representations of these models built using MD simulations (see Materials and Methods). (b) Plots illustrate, for each protein complex, the evolution of the gyration radius Rg (blue dots) during SEC-SAXS elution, the molar mass (red dots) and the forward scattered intensity I(0) (black curve). The dark gray rectangle corresponds to the zone of elution where the successive SAXS curves are rigorously identical. The average of these identical curves was used for analysis.
Figure 5—figure supplement 1—source data 1. SAXS analysis of TDP-43 RRM1–2 fragment alone or bound to GU-repeats (See legend of Figure 5—figure supplement 1a).
Figure 5—figure supplement 1—source data 2. SAXS analysis of TDP-43 RRM1–2 fragment alone or bound to GU-repeats (See legend of Figure 5—figure supplement 1b).
Figure 5—figure supplement 2. Analysis of the kink angle stability.

Figure 5—figure supplement 2.

(a) Time series of the RNA backbone kink angle imposed at G13 by the dimeric assembly of RRM1–2 on (GU)12. The angle was calculated between the phosphate P atoms of U10, G13, and U16. The red line indicates the average angle value of 97.5°. (b) Position of the phosphates used to calculate the kink angle around G13.
Figure 5—figure supplement 2—source data 1. Analysis of the kink angle stability (See legend of Figure 5—figure supplement 2).
Figure 5—figure supplement 3. Interaction of RRM1–2 protein fragment with two different GU-rich oligonucleotides.

Figure 5—figure supplement 3.

Top left panel, superimposition of 1H-15N SOFAST-HMQC spectra of 15N-labeled RRM1–2 bound to (GU)12 (magenta) or bound to 5′-GUGUGAAUGAAUGUGUGUGUGUGU-3’ (AUG12-(GU)6) (green). Top right panel, zoom in on NMR spectra showing unchanged resonances for representative RNA-binding residues as well as residues involved in the protein dimerization (highlighted in red). Bottom panel, Superimposition of calculated (red curve) and experimental (black dots) SAXS curves for the RRM1–2 bound to AUG12-(GU)6. SAXS curves were calculated from all-atoms models using the program GAJOE from the suite EOM. The corresponding χ2 values are indicated. The inset is a 3D representation of the model built using MD simulations.
Figure 5—figure supplement 3—source data 1. SAXS analysis of TDP-43 RRM1–2 fragment bound to AUG12-(GU)6 oligonucleotide (See legend of Figure 5—figure supplement 3).
Figure 5—figure supplement 4. Free energy landscape (FEL) of RRM1–2 dimer in complex with (GU)12 sampled from 110 ns of MD simulation.

Figure 5—figure supplement 4.

FEL is represented using two structural reaction coordinates: the radius of gyration of the system (Rg) and the root mean square deviation (RMSD) with respect to the average structure. The zero energy is at 0 kcal/mol and corresponds to the most stable conformational states. The free energy scale highlights energy differences (0–19 kcal/mol) relative to the global minimum. Radius of gyration and RMSD values are reported in nm. (a) The 3D representation shows ‘valleys’ of low-free energy corresponding to the metastable conformational states of the system, and ‘hills’ that account for the energetic barriers connecting these states. (b) The 2D representation shows the “contour plot” projecting the free-energy surface.
Figure 5—figure supplement 4—source data 1. Ø Free energy landscape (FEL) of RRM1–2 dimer in complex with (GU)12 (See legend of Figure 5—figure supplement 4).
Figure 5—figure supplement 5. Interaction of RRM1–2 protein fragment with GU-repeats.

Figure 5—figure supplement 5.

(a) Superimposition of 1H-15N SEA-HSQC spectra of 15N-labeled RRM1–2 alone (orange) or bound to (GU)6 (turquoise). (b) Superimposition of 1H-15N SEA-HSQC spectra of 15N-labeled RRM1–2 bound to (GU)6 (turquoise) or to (GU)12 (magenta). Zoom in on the superimposed 1H-15N SEA-HSQC spectra of 15N-labeled RRM1–2 bound to (GU)6 (turquoise) or to (GU)12 (magenta). The residues present at RRM1–2 dimerization interface (highlighted in red) are no longer exposed to the solvent.