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

Figure 2. Identification of TDP-43 residues involved in its dimerization on GU-repeats.

(a) NMR spectra of free and bound RRM1–2. Left, superimposition of 1H-15N SOFAST-HMQC spectra of 15N-labeled RRM1–2 in the free (orange) and (GU)6 RNA-bound (turquoise) forms. Residues displaying the largest chemical shift perturbations (CSP) are indicated by arrows. Middle, zoom in on NMR spectra (left) showing the CSPs for some residues (highlighted in red) upon (GU)6 RNA binding. Right, plot of CSPs occurring in RRM1–2 upon (GU)6 RNA binding. The combined CSPs were calculated as reported (Williamson, 2013) and follow the same trajectories as previously published (Lukavsky et al., 2013) for RRM1–2 bound to AUG12 (PDB 4BS2). (b) NMR spectra of monomeric and dimeric forms of RRM1–2 bound to GU-repeats. Left, superimposition of 1H-15N SOFAST-HMQC spectra of 15N-labeled RRM1–2 bound to (GU)6 (turquoise) or (GU)12 (magenta). Right, combined CSPs, observed for monomeric and dimeric couples, plotted and linked to the secondary structures on top. (c) Left, zoom in on NMR spectra (b) showing RRM1–2 residues displaying particular CSPs, resonance disappearing, or peak broadening (in red) as compared to respective residues in RRM1 or RRM2 fragments. Right, all affected residues upon RRM1–2 dimerization are highlighted in red using molecular modelling approaches on RRM1–2 free fragment (see methods). Based on the above comparative NMR study, 28 residues were selected as candidates for mutagenesis approach combined to a detailed cellular and biochemical investigation.

Figure 2—source data 1. NMR data of RRM1-2 in free and bound forms (See legend of Figure 2a,b).

Figure 2.

Figure 2—figure supplement 1. Interaction of RRM1–2 protein fragment with 24 GU-repeats.

Figure 2—figure supplement 1.

Left, superimposition of 1H-15N SOFAST-HMQC spectra of 15N-labeled RRM1–2 bound to (GU)6 (turquoise) or bound to (GU)24 (red). Right, zoom in on NMR spectra showing RRM1–2 residues (highlighted in red) displaying particular CSPs, resonance disappearing or peak broadening upon protein multimerization.
Figure 2—figure supplement 2. Plot showing the intensity ratios calculated between the 15N-labeled RRM1–2 bound to (GU)6 or (GU)12 RNA.

Figure 2—figure supplement 2.

Gray bars correspond to non-assigned residues for which a value equal to 1.0 was arbitrary assigned. Residues located in the long RRM1 loop 3 (a.a.137–146) display low intensities ratios compared to residues located in unstructured domain such as the linker between RRM1 and RRM2 (177–191). In addition, residues around V220 and M202 also display a decreased intensity. Residues selected for the ‘microtubule bench’ assays are indicated by dashed lines.
Figure 2—figure supplement 2—source data 1. NMR data displaying Peak Intensity of RRM1-2 residues in presence of (GU)6 or (GU)12 (See legend of Figure 2—figure supplement 2).