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

Figure 4. Characterization of TDP-43 mutants with an impaired cooperative binding to RNA.

(a) EMSA experiments were performed by using increasing protein concentrations of T141A/G142A, G146A, and Q213A mutants and 10 pmol of a stem-loop DNA to assess multimerization changes on RRM1–2 (as in Figure 1c). Saturated amounts of wild-type RRM1–2 were used as a control (last lane). DNA-protein complexes are pointed out with head-arrows. (b) Plot showing CSPs for RNA-binding residues along RRM1–2 mutants bound to (GU)6 compared to their free forms. In most cases, the binding to (GU)6 RNA provokes CSPs comparable to the wild-type RRM1–2. ΔΔ, ambiguous assignment; Δ, signal vanishing. (c) The binding of RRM1–2 mutants to (GT)6 or (GT)12 oligonucleotides, was monitored by ITC. Whereas the ITC curves of wild type and Q213A are similar (see Figure 1d), the ITC curves related to T141A/G142A and G146A mutants decrease more continuously with less marked plateaus, reflecting an impaired cooperative binding to GT repeats. Lower panel: Kd1/Kd2 ratios obtained from ITC data. T141A/G142A and G146A have lower Kd1/Kd2 ratio values than wild type and Q213A, reflecting an impaired cooperativity. ITC statistics with thermodynamic parameters are indicated in Supplementary file 4. (d) Zoom in on the superimposed 1H-15N SOFAST-HMQC spectra (see full NMR spectra in Figure 4—figure supplement 2) of 15N-labeled RRM1–2 mutants bound to (GU)6 (turquoise) or to (GU)12 (magenta). The residues affected during wild-type RRM1–2 dimerization (see Figure 2b,c) are highlighted (red). Q213A mutant shows the same CSPs as the wild-type protein. However, in the case of T141A/G142A and G146A, we no longer detected the CSPs associated to dimerization.

Figure 4—source data 1. EMSA experiments on T141A/G142A mutant (see legend of Figure 4a).
Figure 4—source data 2. EMSA experiments on G146A mutant (see legend of Figure 4a).
Figure 4—source data 3. EMSA experiments on Q213A mutant (see legend of Figure 4a).
Figure 4—source data 4. NMR data for RNA-binding residues along RRM1–2 mutants bound to (GU)6 compared to their free forms (see legend of Figure 4b).
Figure 4—source data 5. ITC data obtained from the binding of RRM1–2 mutants to (GT)6 or (GT)12 oligonucleotides (see legend of Figure 4c).

Figure 4.

Figure 4—figure supplement 1. Raw calorigrams corresponding to the ITC curves displayed in Figure 4c.

Figure 4—figure supplement 1.

Figure 4—figure supplement 1—source data 1. ITC data obtained from the binding of RRM1–2 mutants to (GT)6 or (GT)12 oligonucleotides (see legend of Figure 4—figure supplement 1).
Figure 4—figure supplement 2. NMR analysis of the interaction of TDP-43 mutant fragments with GU-repeats.

Figure 4—figure supplement 2.

(a) NMR spectra of monomeric and dimeric forms of RRM1–2 mutants (T141/G142, G146A, and Q213A) bound to GU-repeats. In all cases, the superimposition of 1H-15N SOFAST-HMQC spectra of 15N-labeled mutant bound to (GU)6 (turquoise) or (GU)12 (magenta) is shown. (b) Superimposition of 1H-15N SOFAST-HMQC spectra of 15N-labeled wild-type RRM1–2 (turquoise) and mutants (magenta) both bound to (GU)6.
Figure 4—figure supplement 3. Effect of point mutations on the stability of the monomeric and dimeric structures of wild type and mutants of RRM1–2 fragment.

Figure 4—figure supplement 3.

(a) In each case, the RMSD distribution of the protein Cα atoms (top panel) and RNA (middle panel) is shown. The lower panel illustrates the energy distribution of RNA/protein interactions. (b) Overlay of wild-type and mutant 3D structures sampled from the 100 ns MD simulation trajectories. The upper overlay corresponds to the dimeric form and the lower to the monomeric form, bound to (GU)12 and (GU)6, respectively.
Figure 4—figure supplement 3—source data 1. Effect of point mutations on the stability of the monomeric and dimeric structures of wild type and mutants of RRM1–2 fragment (see legend of Figure 4—figure supplement 3).